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Volume 6 Number 1 Jun 2010

Editorial

Salleh Mohd. Nor and Mohinder Singh 3

Comparison of water and gastrografin as an oral contrast medium for

abdominopelvis computed tomography

K. C. Kanaga, S. E. Laila, P. Y. Teh and J. Shukriah 5

Effect of colchicine on tissue culture derived plants of Zingiber officinale Rosc.

and Zingiber officinale var. rubrum Theilade

Lindayani, K. Norzulaani, H. Ibrahim and Noorsaadah Abd Rahman 11

Diversity and abundance of Dacinae fruit flies (Insecta: Diptera: Tephritidae) in Chini 2,

Runchang and Sungai Bebar, Pahang, Peninsular Malaysia

Hoi Sen Yong, Y. F. Ng and Phaik Eem Lim 17

The mammal fauna of Pulau Singa Besar, Langkawi, Kedah, Peninsular Malaysia

Lim Boo Liat, Norhayati Ahmad, O. Noor Alif Wira, Shahrul Anuar Md. Sah 23

Antiproliferative and antioxidant properties of leaf extracts of Pereskia bleo (Cactaceae) and

their ability to limit natural and oxidant-induced apoptotic cell death

Hui Meng Er, Hong Lim Lee, Ammu Kutty Radhakrishnan 31

Abdominal colour polymorphism in female Asian Golden Web Spider Nephila antipodiana

(Araneae: Nephilidae)

Yong Hoi Sen, Rosli Hashim, Daicus Belabut and Lim Phaik Eem 39

Scaled down operation of the United Nations University/International Centre for

Theoretical Physics Plasma Focus Facility (UNU/ICTP PFF) as an extreme ultraviolet source

Rattachat Mongkolnavin, Prajya Tangitsomboon and Chiow San Wong 43

An atmospheric pressure non-thermal plasma jet in nitrogen for surface modification of polyethylene

D. P. Subedi, R. B. Tyata, A. Shrestha, D. Baral, D. K. Madhup and C. S. Wong 49

Compressed natural gas (CNG) cylinder testing and data evaluation using

acoustic emission technique

Tonphong Kaewkongka, Jirapong Lim and Suparerk Sirivedin 53

Prompt gamma neutron activation analysis (PGNAA) of hydrocarbons:

A Monte Carlo study with GEANT4

Lam YiHua, Wong Chiow San and Kurunathan Ratnavelu 59

Jostt vol 6.indd 1 7/22/10 10:08:24 PM


JOURNAL OF SCIENCE AND TECHNOLOGY IN THE TROPICS

INSTRUCTIONS TO CONTRIBUTORS

JOSTT is a multi-disciplinary journal. It publishes original

research articles and reviews on all aspects of science

and technology relating to the tropics. All manuscripts

are reviewed by at least two referees, and the editorial

decision is based on their evaluations.

Manuscripts are considered on the understanding that

their contents have not been previously published, and

they are not being considered for publication elsewhere.

The authors are presumed to have obtained approval

from the responsible authorities, and agreement from all

parties involved, for the work to be published.

Submission of a manuscript to JOSTT carries with

it the assignment of rights to publish the work. Upon

publication, the Publishers (COSTAM and ASM) retain the

copyright of the paper.

Manuscript preparation

Manuscripts must be in English, normally not exceeding

3500 words. Type double spaced, using MS Word, on one

side only of A4 size with at least 2.5 cm margins all round.

Number the pages consecutively and arrange the items

in the following order: title page, abstract, key words, text,

acknowledgements, references, tables, figure legends.

Title page

Include (i) title, (ii) names, affiliations and addresses of all

authors, (iii) running title not exceeding five words, and

(iv) email of corresponding author.

Abstract and key words

The abstract, not more than 250 words, should be

concise and informative of the contents and conclusions

of the work. A list of not more than five key words must

immediately follow the abstract.

Text

Original research articles should be organized as follows:

Introduction, Materials and Methods, Results, Discussion,

Acknowledgement, References. The International

System of Units (SI) should be used. Scientific names and

mathematical parameters should be in italics.

References

References should be cited in the text as numbers

enclosed with square [ ] brackets. The use of names in

the text is discouraged. In the reference section, the

following examples should be followed:

1. Yong H.S., Dhaliwal S.S. and Teh K.L. (1989)

A female Norway rat, Rattus norvegicus,

with XO sex chromosome constitution.

Naturwissenschaften 76: 387-388.

2. Beveridge W.I.B. (1961) The Art of Scientific

Investigation. Mercury Book, London.

3. Berryman A.A. (1987) The theory and

classification of outbreaks. In Barbosa P. and

Schultz J.C. (eds.) Insect outbreaks pp. 3-30.

Academic Press, San Diego.

Tables

Tables should be typed on separate sheets with short,

informative captions, double spacing, numbered

consecutively with Arabic numerals, and do not contain

any vertical lines. A table should be set up to fit into the

text area of at most the entire page of the Journal.

Illustrations

Black-and-white figures (line drawings, graphs and

photographs) must be suitable for high-quality

reproduction. They must be no bigger than the printed

page, kept to a minimum, and numbered consecutively

with Arabic numerals. Legends to figures must be typed

on a separate sheet. Colour illustrations can only be

included at the author’s expense.

Proofs and reprints

Authors will receive proofs of their papers before

publication. Order for reprints must be made when

returning the proofs.

Submission

Manuscripts (including all figures but not original

artwork), together with a CD version of the text, should

be submitted to:

The Editorial Office

Journal of Science and Technology

in the Tropics

Academy of Sciences Malaysia

902-4 Jalan Tun Ismail

50480 Kuala Lumpur, Malaysia

E-mail: jostt@akademisains.gov.my

JOSTT is listed in Scopus

Jostt vol 6.indd 2 7/22/10 10:08:24 PM


Journal of Science and Technology in the Tropics (2010) 6: 3

Editorial

PUBLISH OR PERISH

When one chooses a scientific career, it becomes a norm to publish. One has to disseminate one’s

findings and one’s work. It is part of the challenge of being a scientist and it is expected of a scientist.

Some institutions set target for scientists to publish at least two scientific papers in a refereed journal

a year. Such targets are good as there is a propensity for scientists in general not to publish.

There are many forms of publications. The usual is a scientific journal and most professions or

institutions have their own dedicated journals to provide a conduit for their scientific staff to publish.

However, the growing concerns on the environment and the destruction of forests have been used

as an argument to choose electronic publications and the widespread use of the internet allows this.

However, among the more senior scientists, the preference is still the written media. Publishing in

peer-reviewed journals should be encouraged as this will provide a more international audience. The

impact factor of the journal should also be taken into consideration. The number of times that an

article is cited or the citation index is also an indicator of the readership of the article.

The other issue often debated is proceedings of seminars and conferences. Conferences, workshops

and seminars provide excellent channels for the dissemination of research findings and ideas, and

proceedings of such meetings are critically important to record the presentations that would be lost

otherwise.

There is an increasing propensity for joint authorship of papers. While this is inevitable considering

that research is seldom undertaken by an individual but rather by a team, the tendency for the head of

the organization to be the main author should not be encouraged unless he or she was really the main

researcher involved in the project. The main scientist that undertook the major part of the research

and who wrote the article must be the main author.

Some disciplines are more amenable to publications. For example, in taxonomic research it is

much easier to publish when the scientist has completed the study of a certain species of plant or

animal or microorganism. However, in some other disciplines, it is much more difficult and time

consuming. Nonetheless, the key to good research is innovation and such innovative discoveries or

ideas should be published.

Finally there is the fundamental issue of scientific writing. Many scientists have never been taught

the art or science of scientific writing. It is proposed that every young scientist undergo a course on

scientific writing early in their career as this will give them confidence and provide them a good

foundation to publish their findings later.

Dr. Salleh Mohd. Nor and Dr. Mohinder Singh

Co-Chairman, Editorial Board

Jostt vol 6.indd 3 7/22/10 10:08:25 PM


Jostt vol 6.indd 4 7/22/10 10:08:25 PM


Journal of Science and Technology in the Tropics (2010) 6: 5-9

Comparison of water and gastrografin as an oral contrast medium for

abdominopelvis computed tomography

K. C. Kanaga 1 , S. E. Laila1, P. Y. Teh 1 and J. Shukriah 2

1 Diagnostic Imaging and Radiotherapy Programme, Faculty of Allied Health Sciences,

Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd Aziz, 50300 Kuala Lumpur, Malaysia

(Email: kanagakkc@yahoo.com)

2 Diagnostic Imaging Department, Kuala Lumpur Hospital, Jalan Pahang, 50300 Kuala Lumpur, Malaysia

Received 25-01-2010; accepted 06-04-2010

Abstract The aim of this study was to determine if water was comparable to gastrografin as an oral contrast

medium in defining anatomic details of abdominopelvis computerized tomography. A total of 98 patients referred

to the Diagnostic Imaging Department, Hospitak Kuala Lumpur from January to April 2009 for abdominopelvis

CT with intravenous contrast enhancement were studied using Siemens Somatom Sensation 16 scanner. The

results showed that water, with superior distension and delineation capabilities of the gaster and bowel, was

comparable to gastrografin as an oral contrast medium.

Keywords gastrografin – water – contrast medium – quantitative – qualitative

INTRODUCTION

Multidetector computed tomography (CT), with high

speed multiplanar imaging has led to a paradigm

shift in abdominopelvis imaging. CT is also capable

of producing thinner slices within short scan time

and fast intravenous infusion of contrast material

[1]. With this new technique, the use of highattenuation

positive contrast medium to outline

the stomach and small bowel may degrade image

quality especially in procedures requiring maximum

intensity reconstruction [2]. Besides, adequate mural

enhancement for diagnosis of abnormalities may

be absent due to the almost non-existent difference

between enhanced bowel wall and high attenuation

intraluminal content [3]. An oral contrast medium

should have a high distension capability that delivers

optimal differentiation between lumen and bowel

wall. Excellent mixing of contrast medium and

the contents of gastro-intestinal tract may lead to

consistent pacification and accurate interpretation of

the images [4].

Neutral contrast agent produces improved image

quality and facilitates the diagnosis of bowel wall

abnormalities. It has been proven to be effective in the

diagnosis of Crohn’s disease [5], neoplasms [6] and

bowel-ischemia [7]. Distension and delineation of the

stomach and duodenum in imaging of pancreatic and

biliary disease has been demonstrated using neutral

contrast [8]. The most widely used neutral contrast

agent is water as it has shown excellent result in the

upper gastrointestinal tract; however its clinical use

for the distal parts of the small bowel is limited due to

its rapid absorption [9].

Studies have been done to investigate a negative

contrast agent that can be used routinely for all

types of CT abdominopelvis as an alternative for the

positive oral contrast agent but the results obtained

have been inconclusive [10, 11].

This study was designed to determine if water

was comparable to gastrografin as an oral contrast

medium in defining anatomic details. The hypothesis

of our study was water would be as effective or even

better then gastrografin for abdominopelvis CT.

MATERIALS AND METHODS

A total of 98 patients referred to the Diagnostic

Imaging Department, Hospital Kuala Lumpur from

January to April 2009 for abdominopelvis CT with

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Journal of Science and Technology in the Tropics (2010) 6: 5-9

intravenous contrast enhancement were studied

using Siemens Somatom Sensation 16 scanner. The

study was approved by the Ethics Committee of the

Ministry of Health Malaysia.

Patients were divided into two groups of 49

patients each. The patients were randomly selected

by predefined inclusion and exclusion criteria to

participate in either of the two groups. The clinical

indications related to liver (30%), pancreas (30%),

renal (20%) and GIT (20%) for both groups did not

differ statistically with regard to their distribution (χ 2

test).

All patients were required to fast for at least 6

hours. Water or gastrografin (500 mL) was given at

60, 30 minutes and immediately before scanning. For

gastrografin 10 mL was diluted into 500 mL solution.

A scout image from xiphoid to the symphysis was

performed using a 16-detector row CT scanner

(Siemens, Somaton Sensation 16), followed by a

intravenous contrast bolus administration of Iopamiro

300 with a rate of 3 ml/s. Acquisition of scans was

obtained using the same protocol of 5-mm transverse

sections.

Two experienced radiologists independently

reviewed the images from each examination in

transverse and coronal planes on the 3 megapixel

workstation. The radiologists were blinded to the

clinical history of the patients. An incremental threepoint

scale (0 = worst, 0.5 = moderate, 1 = best) was

used to rate images from each examination for bowel

distension, homogeneity of the lumen, delineation

of the bowel wall, presence of artifacts and overall

image quality.

Overall bowel distension was graded from totally

collapsed (0) to maximal distension (1); homogeneity

of the lumen from massively inhomogeneous (0) to

completely homogeneous (1). The delineation of

the bowel wall was rated twice from indiscernible

(0) to clearly visible (1), initially against the

intraluminal contrast and then against the surrounding

extraintestinal tissue. The presence of artifacts was

rated as no artifacts (0) to serious image degradation

due to artifacts (1). Finally, overall image quality was

assessed from unreadable (0) to perfect (1).

To support the qualitative evaluation, additional

quantitative measurements were performed. The

maximum cross-sectional diameter of the antrum

of the stomach, horizontal part of the duodenum,

proximal jejunum, and terminal ileum were measured

perpendicular to the axis of the lumen using the

outer margins of the intestinal wall for each patient.

Attenuation in Hounsfield units (HU) of the lumen

and gastrointestinal wall was measured at the

same levels of the stomach, duodenum, jejunum,

and terminal ileum. Attenuation of the lumen was

measured by placing a region of interest (ROI)

within a well-distended segment of the small bowel

section. Attenuation of the wall was measured by first

zooming into the image section until the wall was

clearly visible, and then placing an ROI (minimum

diameter of 2 mm) over the bowel wall.

Mann-Whitney U-test was used to perform

statistical analysis of the differences in median scores

between water and gastrografin groups regarding

bowel distension, homogeneity of the lumen,

differentiation of the bowel wall against luminal

content and surrounding fat, the presence of artifacts

and overall image quality. Differences were considered

significant if p < 0.05; inter-observer agreement was

evaluated using weighted kappa statistics where a

kappa statistics > 0.75 was considered as excellent

agreement, 0.4-0.75 as fair to good agreement, and <

0.4 as poor agreement. Values near zero or less than

zero reflected only chance agreement.

For the quantitative analysis, the differences in

maximum diameters, HU values for the bowel lumen,

and contrast values between the neutral oral contrast

groups were compared using t-test; differences were

considered significant if p < 0.05

RESULTS

All patients tolerated the administration of oral

contrast medium. There was no vomiting, diarrhoea,

abdominal pain or allergic reactions to both the oral

contrast media. All patients drank the designated

amount of fluid within the given time. The images

were of diagnostic quality to be evaluated by two

independent radiologists with five years of experience.

Kappa analysis showed the inter-observer agreement

in the excellent category (k = 0.762).

The qualitative results were summarized in Figure

1. Delineation of the bowel wall from inside to outside

was significantly better using water compared to

gastrografin (p < 0.05). As for all the other criteria

there was no significant difference between water

and gastrogafin. The quantitative measurements are

summarized in Table 1. Diameters of the gaster and

Jostt vol 6.indd 6 7/22/10 10:08:26 PM



Location Oral contrast

Gastric

Duodenum

Jejunum

Iliem

Figure 1. Qualitative evaluation of abdominopelvis

CT images, water vs gastrografin

Table 1. Quantitative evaluation of gastrointestinal tract,

gastrografin vs water.

bowel were significantly better using water compared

to gastrografin (p < 0.05) while the other criteria

showed no significant difference using t-test and

Mann-Whitney U-test .

DISCUSSION

Diameter

(cm)

Attenuation

of bowel

wall (HU)

Attenuation

of bowel

lumen (HU)

Difference

wall to

lumen

Gastrografin 9.81 128.67 100.24 -28.43

Water 10.62 -7.46 52.56 60.02

p>0.05 p


8

JJournal of Science and Technology in the Tropics (2010) 6: 5-9

Figure 4. CT images of abdominopelvis with water

as the contrast medium.

reconstruction and computer workstations for

anatomical reconstruction have made it possible to

interpret every structure in abdominal CT studies,

including organs, vessels, bones and also bowel, in

a single examination (Fig. 4). Besides, ability of CT

that scans at a shorter time greatly eliminates artifacts

caused by peristaltic and respiratory movement. The

results were consistent with previous studies that

stated that positive oral contrast caused more streak

artifacts compared to water [11]. CT images can also

be reconstructed to get detail visualization of bowel

wall [18].

Subtle changes in the bowel wall and its

surrounding fat can be masked with the use of

positive oral contrast agent like gastrografin. The

reasons for this are enhanced bowel wall may have

the same attenuation as the positive contrast of

the lumen and high densities often occur due to

heterogeneous intraluminal distribution, leading to

artifacts with reduced visualization of the bowel wall

1. Foley W. D. and Kerimoglu U. (2004) Abdominal

MDCT; liver, pancreas, and biliary tract. Semin

Ultrasound CT MR 25: 122-144.

2. Horton K. M., and Fishman E. K. (2003) the current

status of multidetector row CT and three-dimensional

imaging of the small bowel. Radiol Clin North Am 41:

199-212.

3. Raptopoulos V., Davis M. A., Davidoff A., Karellas

A., Hays D., D’Orsi C. J. and Smith E. H. (1987)

Fat-density oral contrast agent for abdominal CT.

Radiology 164: 653-656.


REFERENCES

and surrounding fat [16]. This may be the reason

for increasing interest in the use of low attenuation

contrast for a wider range of indications. Water is the

primary choice; advantages include low cost, wide

availability, natural and safe for anyone without any

complications [7].

Water showed significantly better contrast between

bowel wall and lumen and for other criteria in

qualitative analysis, including homogeneity in lumen,

delineation of bowel wall and overall quality image,

consistent with other previous studies. Combination

of water that acts as negative oral contrast agent and

intravenous contrast agent that acts as positive contrast

agent give a better appreciation of mural detail.

Mucosal folds can be visualized clearly and actual

bowel wall thickness can be determined. However,

the limitation of using water as an oral contrast agent

is that the patient needs to consume a large quantity

of water, especially for those in-patients who are very

ill. Some of them even vomit after trying to take more

water. Previous study reported that there were cases

of water toxicity after consuming large amount of

water by patients who had chronic renal failure and

congestive heart failure [7]. Water is not a suitable

oral contrast for these types of patients.

In summary, this study showed that water was

comparable to gastrografin as an oral contrast

medium. Qualitative evaluation showed significantly

superior bowel distension and contrast between bowel

wall and lumen. Besides, quantitative measurements

showed better distension of gaster and bowel. Water

is therefore a better choice as oral contrast medium

since it is inexpensive and safe to consume.

4. Turetschek K., Schober E., Wunderbaldinger P.,

Bernhard C., Schima W., Puespoek, A., Vogelsang

H., Moeschl P. and Mostbeck G. (2002) Findings at

Helical CT-Enteroclysis in Symptomatic Patients

With Crohn Disease: Correlation With Endoscopic

and Surgical Findings. Journal of Computer Assisted

Tomography 26: 488-492.

5. Horton K. M., and Fishman E. K. (2004)

Multidetector-row computed tomography and

3-dimensional computed tomography imaging of

small bowel neoplasms: current concept in diagnosis.

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Journal of Computer Assisted Tomography 28: 106-

116.

6. Horton K. M., and Fishman E. K. (2001) Multidetector

row CT of mesenteric ischemia: can it be done?

Radiographics 21: 1463-1473.

7. Winter T. C., Ager J. D., Nghiem H. V., Hill R. S.,

Harrison S. D. and Freeny P. C. (1996) Upper

gastrointestinal tract and abdomen: water as an orally

administered contrast agent for helical CT. Radiology

201: 365-370.

8. Hori S., Tsuda K., Murayama S., Matsushita M.,

Yukawa K. and Kozuka T. (1992) CT of gastric

carcinoma: preliminary results with a new scanning

technique. Radiographics 12: 257-268.

9. Spilde J., Lee F. T., Chosy S. G., Krupinski E. A.,

Kuhlman J. E. and Yandow M. R. (1999) Evaluation

of an experimental low-attenuation gastrointestinal

contrast agent for CT imaging of intestinal ischemia

in an animal model. Acad Radiol 6: 94-101.

10. Thompson S. E., Raptopoulos V., Sheiman R. L.,

McNicholas M. M. J. and Prassopoulos P. (1999)

Abdominal helical CT: Milk as low-attenuation oral

contrast agent. Radiology 211: 870-875.

11. Matsuoka Y., Masumoto T., Koga H., Suzuki K.,

Ushimi T., Terada H., Tamura A., Yokoyama Y., Abe

K. and Kamata N. (2000) Positive and negative oral

contrast agents for combined abdominal and pelvic

helical CT: first iodinated agent and second water.

Radiation Medicine 18: 213-216.

12. Berther R., Patak M. A., Eckhardt B., Erturk S. M.

and Zollikofer C. L. (2008) Comparison of neutral

oral contrast versus positive oral contrast medium in

abdominal multidetector CT. Eur Radiol. 18: 1902-

1909.

13. Balfe D. M., Koehler R. E., Karstaedt N., Stanley R.

J. and Sagel S. S (1981) Computed tomography of

gastric neoplasm. Radiology 140: 431-436.

14. Komaki S. and Toyoshima S. (1983) CT’s capability

in detecting advance gastric cancer. Gasrointest Radiol

8: 307-313.

15. Macari M. and Balthazar E. J (2001) CT of bowel wall

thickening: Significance and pitfalls of interpretation.

AJR 176: 1105-1116.

16. Horton K. M., Fishman E. K. (1998) The current

status of multidetector row CT and three-dimensional

imaging of the small bowel. Radiol Clin North Am

41:199–212.

17. Halvorsen R. A. (2006) MDCT: A practical research.

MDCT of abdominal trauma. pp. 185-195. Springer,

Milan.

18. Gossios K. J., Tsianos E. V., Demou L. L., Tatsis C. K.,

Papakostas V. P., Masalas C. N., Merkouropoulos M.

C. and Kontogiannis D.S. (1991) Use of water or air as

oral contrast media for computed tomographic study

of the gastric wall: comparison of two techniques.

Gastrointest Radiol. 16: 293-297.

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Journal of Science and Technology in the Tropics (2010) 6: 5-9


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Journal of Science and Technology in the Tropics (2010) 6: 11-16

Effect of colchicine on tissue culture derived plants of Zingiber officinale Rosc.

and Zingiber officinale var. rubrum Theilade

Lindayani 1 , K. Norzulaani 2 , H. Ibrahim 2 and Noorsaadah Abd Rahman 3

1 Department of Food Technology, Faculty of Agricultural Technology, Soegijapranata Catholic University,

Pawiyatan Luhur IV/1 Bendan Duwur, Semarang 50234, Indonesia

2 Institute of Biological Sciences, 3 Department of Chemistry, Faculty of Science, University of Malaya,

Lembah Pantai, 50603 Kuala Lumpur, Malaysia

(Email: lindayani@unika.ac.id)

Received 04-01-2010; accepted 01-02-2010

Abstract The aim of this study was to assess the effect of colchicine on the morphology and histology of

gingers in view of polyploidisation. Rhizomes of ginger were treated separately with three concentrations of

colchicine (0.5%, 1.0% and 2.0% w/v) for 30, 60 and 120 minutes. For morphology study, the fresh weight

of rhizome (FWRH), length of root (RLEN) and plant height (PH) were measured. Histological analysis was

done on shoot buds and rhizomes. All the parameters tested gave significant result. An increasing trend was

observed with colchicine concentration up to 1.0%, but decreased at 2.0%. Histological analysis showed that

the cell size of the shoot tip of Zingiber officinale Rosc. treated with colchicine (2.0%) for 120 minutes was 1.5

times larger than the control. For Zingiber officinale var. rubrum Theilade, the cell size was similar for treated

and control shoot tips. Over all this study showed that polyploidisation might occur with colchicine treatment

of various concentration and incubation time.

Keywords plant morphology – histology – polyploidisation – ginger

INTRODUCTION

Ginger, Zingiber officinale Rosc (Zingiberaceae), has

been used as a spice both in the East as well as in

the West since time immemorial [1]. In Peninsular

Malaysia there are at least three local races, namely

halia betul (true ginger), halia bara (red ginger) or

alternatively halia padi, and halia udang [2]. Both

halia bara and halia padi are distinguished from

halia betul from their small rhizomes. Halia udang is

probably extinct but halia bara differs slightly from

halia padi by its externally red rhizome. Halia bara

and halia padi are more pungent than the normal

ginger and are mainly used in traditional medicine

[2,3]. Pharmacological studies have shown that

ginger rhizomes are effective for the recovery of

intestinal disorder [4] and salivary secretion [5], for

stimulating the vasomotor and respiratory centres,

and for lowering serum and hepatic cholesterol levels

[6,7].

Apart from the normal mitotic processes, polyploidy

can be induced by treatment with colchicine [8-10].

The in vitro induction of polyploids with colchicine

has been reported in many plant species [11-15]. The

increase in the ploidy level could occur relatively

easily and may lead to an overall enlargement of plant

organs, for example cells, stomata, leaves, flowers,

fruits and seeds [8].

Tetraploid gingers (Z. officinale) strains “4x

Kintoki”, “4x Sanshu” and “4x Phillippine Cebu 1”

have been produced by soaking shoot tip explants

in a colchicine solution (0.2%, w/v). The induced

tetraploid gingers were much bigger in plant and

rhizome size than the diploids [14]. In addition,

the tetraploid ginger had higher pollen fertility and

germination rates than the diploids. However, seed

settings in the tetraploid ginger have not yet been

reported [16].

Colchicine prevents spindle formation at prophase,

precludes a nuclear mitosis, delays chromosomal

separation, inhibits daughter nuclei, and effectively

blocks the cleavage processes. Hence, when root tips

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Journal of Science and Technology in the Tropics (2010) 6: 11-16

or other growing plant parts were placed in appropriate

concentration of colchicine, the chromosomes of the

treated cells duplicated without spindle formation

and the cytoplasmic phase of cell division would not

occur [17].

In general, colchicine is used as an antimitotic

agent in plants. It influences the duplication of the

number of chromosome. The effect of colchicine

could be determined through the plant morphology.

The present study was carried out to examine the

effect of colchicine on the morphology and histology

on tissue culture derived ginger plants Zingiber

officinale and Zingiber officinale var. rubrum.

MATERIALS AND METHODS

Plant Material

The rhizomes of Z. officinale and Z. officinale var.

rubrum used in this study were obtained from the

local wet market. The specimens were authenticated

by Halijah Ibrahim from the Institute of Biological

Sciences, University of Malaya.

Application of colchicine

Colchicine solutions were cooled after autoclaving

and were freshly prepared before use. Fourteen to

Table 1. Combinations of treatment with colchicines

concentration (C) and incubation time (T) used for

in vitro derived shoot buds of ginger.

Concentration of

Colchicines (%)

CO (0.0)

C1 (0.5)

C2 (1.0)

C3 (2.0)

Incubation time

(minutes)

TO (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

Number of

Shoot bud

16

16

16

16

14

14

14

14

14

14

14

14

14

14

14

14

sixteen shoot bud pieces were used for each

treatment combination (Table 1). These buds were

soaked in sterile distilled water (control), 0.5%

(w/v), 1.0% (w/v) or 2.0% (w/v) aqueous colchicine

solution in a 250-mL Erlenmeyer flask placed on an

orbital shaker (90 rpm) at 24±2°C. The shoot buds

were incubated according to the selected time (Table

1). Following that, each bud was dried on sterile

filter paper and subsequently placed in MS semisolid

initiation media under a 16 hours photoperiod at

3500 lx using white fluorescent tubes. The

temperature was maintained at 24±2°C in the growth

room. After about five months (5 th subculture),

the surviving colchicine-treated plantlets were

transferred to sterile vermiculite placed in plastic

polybags (3″ x 6″) and kept in a growth room under

a 16:8-h light:dark photoperiod at 24±2°C for 4

months before transferring to the nursery.

Growth maintenance of colchicine treated plants

After the plantlets had rooted and reached 3-5 cm

in height with well expanded leaves, they were

transplanted into pots filled with soil mix consisting

of sand:peat:top soil (3:2:1), vermiculite and fertilizer

(N:P:K at the ratio of 15:15:15) after washing off the

agar with tap water. To avoid fungal infection, the soil

mix in the pot was drained with 1% (w/v) Thiram ®

over night before planting. For pest control, plants

were sprayed with 1.0 mL of Plusbon 250 ® diluted

with 5 L water. Plants were irrigated daily and 1.0 g

of foliar fertilizer (Agrospray 63 ® ) diluted in 5 L tap

water was sprayed monthly for supplement.

Measurement of plant morphology

The plants were monitored every month. They

were maintained in the nursery for one year before

harvesting and analysis. For each treatment, fresh

weight of rhizome (FWRH) and plant height (PH)

were measured. Length of the root (RLEN) was

measured randomly by using Image-Pro ® Express

Version 4.5 (Media Cybernetics).

Histological analysis

The shoot buds were fixed in FAA (formalin, acetic

acid glacial, ethyl alcohol: 5:5:90), then progressively

dehydrated in ethanol series, and finally infiltrated in

paraffin. Longitudinal sections (10-20 µm thick) of

paraffin-embedded materials were obtained using a

Jostt vol 6.indd 12 7/22/10 10:08:34 PM


otary microtome. The sections were stained with

a mixture of 1% (v/v) safranine and 1% (v/v) fast

green and mounted with Canada Balsam [18]. Size

of cells was measured using a micrometer eyepiece

where 0.01 mm equals to 5 mm (under x100 and x400

magnification). This work was done at the laboratory

of plant histology, Biological Faculty, University of

Gadjah Mada, Yogyakarta, Indonesia.

Statistical analysis

Analysis of variance (ANOVA), performed with the

program SAS (SAS ® proprietary software Release

6.02), of factorial design was used to test the effect of

the concentrations of colchicine and incubation times.

Duncan Multiple Range Test (DMRT) at p


14

Journal of Science and Technology in the Tropics (2010) 6: 11-16

Plant height measured for Z. officinale (V1) showed

the highest reading (48.24 cm) for treatment C1T1

compared to the other treatments. C1T1 treatment


Figure 2. Rhizome of Zingiber officinale, V1, C0T0

(1) and Zingiber officinale var. rubrum, V2, C0T0

(2) obtained from control; and rhizome of Zingiber

officinale, V1, C3T2 (3) and Zingiber officinale var.

rubrum, V2, C1T1 (4) after treatment. C0T0 = Control;

C3T2 = colchicine conc. 2%, incubation time 60 min;

C1T1 = Colchicine conc. 0.5%, incubation time 30 min.


for Z. officinale var. rubrum (V2) exhibited similar

result for the control (43.38 cm) and treated group

(43.16 cm) (Table 3.). Roots for Z. officinale (V1)

were shorter compared to Z. officinale var. rubrum

(V2). The best result for Z. officinale was produced

by treatment C1T1 while for Z. officinale var. rubrum

the longest root measurement was obtained when the

shoot bud was briefly dipped in treatment C1T0.

To date, there are no reports on the morphology of

ex vitro colchicine treated plants as described at this

study. Based on this study, an increasing trend for fresh

Table 3. Fresh weight of rhizome (FWRH, g), PH,

plant height (PH, cm) and root length (RLEN, cm) from

combination of variety, concentration of colchicine and

incubation time after transferring to the nursery. V, Variety

(V1, Zingiber officinale; V2, Zingiber officinale. var.

rubrum ); C, concentration of colchicine; T, incubation time.

Different alphabets show significant differences (p


weight of rhizome, plant height and root length was

observed with increasing colchicine concentration up

to 1.0% but decreased at 2.0%. For incubation time,

results were not consistent. Based on morphological

characteristics (fresh weight of rhizome, plant height

and root length) for treated plants, significant results

were found in all the data. When colchicines was

used, various organs of tetraploid gingers become

huge in appearance i.e. plant height, the size of leaves

and rhizomes, compared to diploids [14].

Histological analysis of shoot tips

Preliminary results showed that the cell size of the

shoot tip treated with colchicine (2.0%, C3) and

incubation time (T3, 120 minutes) was 1.5 times

larger than the control (C0) for sample V1 (Fig. 3).

For sample V2, the treated and control shoot tips

had similar cell size (Fig. 4).

From this preliminary histological analysis, the

effects of colchicine concentration were observed for

sample V1, but the result obtained did not confirm

or prove that polyploidisation was induced by the

treatments. Future work on quantitative analysis is

needed.

CONCLUSION

The effect of colchicine treatment on Z. officinale

and Z. officinale var. rubrum could be observed in

the morphology and histology of the treated plants.

An increase in the fresh weight of the rhizomes of

the treated plants was observed as compared to

the control. However, the results from qualitative

histological studies were inconclusive.


Figure 3. Micrographs of a section of the shoot tips of Zingiber officinale (V1). C0 (control) and C3 (treatment

with colchicine concentration 2.0% and incubation time 120 minutes).


Figure 4. Micrographs of a section of the shoot tips of Zingiber officinale var. rubrum (V2). C0 (control) and C1

(treatment with colchicine concentration 0.5% and incubation time 30 minutes).

Jostt vol 6.indd 15 7/22/10 10:08:40 PM

15

Journal of Science and Technology in the Tropics (2010) 6: 11-16


16

Journal of Science and Technology in the Tropics (2010) 6: 11-16

1. Larsen K., Ibrahim H., Khaw S.H. and Saw L.G.

(1999) Gingers of Peninsular Malaysia and

Singapore. Natural History Publications (Borneo),

Kota Kinabalu.

2. Holttum R.E. (1950) The Zingiberaceae of the Malay

Peninsular. The Garden’s Bulletin Singapore 13:

1-250.

3. Purseglove J.W. (1972) Zingiberacea. In Purseglove

J.W. (Ed.) Tropical crops monocotyledons 2 pp. 519-

540. Longman Group.

4. Krishnamurthy K. and Sreenivasamurthy V. (1956)

Garlic. Bull. Centr. Food Technol. Res. Inst. Mysore.

5, 264-267.

5. Sinha K.P., Sud S.C. and Bahga H.S. (1974) Physiology

of digestion in ruminants 3 salivary secretion. Effect

of autonomic drugs. Indian Veterinary Journal 51:

15-19.

6. Gujral S., Bhumra H. and Swaroop M. (1978) Effect

of ginger (Zingiber officinale Roscoe) oleoresin on

serum and hepatic cholesterol levels in cholesterol

fed rats. Nutrition Report International 17: 183-189.

7. Newall C.A., Anderson L.A. and Phillipson J.D.

(1996) Herbal Medicines A guide for health-care

professionals. The Pharmaceutical Press, London.

8. Van Harten A.M. (1998) Mutation breeding: theory

and practical applications. Cambridge University

Press.

9. Rey H.Y., Sansberro P.A., Collavino M.M.,

Daviña J.R., Gonzàles A.M. and Mroginski

10.

L.A. (2002) Colchicine, trifluralin, and oryzalin

promoted development of somatic embryos in Ilex

paraguariensis (Aquifoliaceae). Euphytica 123: 49-

56.

Petersen K.K., Hagberg P. and Kristiansen K. (2003)

Colchicine and oryzalin mediated chromosome

doubling in different genotypes of Miscanthus

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12.

13.

sinensis. Plant Cell, Tissue and Organ Culture 173:

137-146.

Gao S.L., Zhu D.N., Cai Z.H. and Xu D.R. (1996)

Autotetraploid plants from colchicines-treated bud

culture of Salvia miltiorrhiza Bge. Plant Cell, Tissue

and Organ Culture 47: 73-77.

Ma Y., Chen D.H. and Chen J. (1997) Amphidiploid

induction from diploid rose interspecific hybrids.

Horticulture Science 32: 292-295.

Chakraborti S.P., Vijayan K., Roy B.N. and Qadri

S.M.H. (1998) In vitro induction of tetraploidy in

mulberry (Morus alba L.). Plant Cell Reports 17:

799-803.

14. Adaniya S. and Shirai D. (2001) In vitro induction

15.

16.

of tetraploid ginger (Zingiber officinale Roscoe)

and its pollen fertility and germinability. Scientia

Horticulturae 88: 277-287.

Chauvin J.E., SouchetC., Dantec, J.P. and Ellissèche

D. (2003) Chromosome doubling of 2x Solanum

species by oryzalin: method development and

comparison with spontaneous chromosome doubling

in vitro. Plant Cell, Tissue and Organ Culture 73: 65-

73.

Adaniya S. (2001) Optimal pollination environment

17.

of tetraploid ginger (Zingiber officinale Roscoe)

evaluated by in vitro pollen germination and pollen

tube growth in styles. Scientia Horticulturae 90: 219-

226.

Eigsti O.J. and Dustin P. (1957) Colchicine in

agriculture, medicine, biology, and chemistry. The

Iowa State College Press, Ames, Iowa. U.S.A.

18. Johansen D.A. (1940) Plant microtechnique.

McGraw-Hill, New York.

19. Gomez K.A and Gomez A.A. (1984) Statistical

procedures for agricultural research. Second Edition.

John Wiley & Sons.

Jostt vol 6.indd 16 7/22/10 10:08:41 PM


Journal of Science and Technology in the Tropics (2010) 6: 17-21

Diversity and abundance of Dacinae fruit flies (Insecta: Diptera: Tephritidae) in

Chini 2, Runchang and Sungai Bebar, Pahang, Peninsular Malaysia

Hoi Sen Yong1 , Y. F. Ng2 and Phaik Eem Lim1,3 1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

(E-mail: yong@um.edu.my)

2Centre for Insect Systematics, School of Environmental and Natural Resource Sciences,

Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 4300 Bangi,

Selangor D.E., Malaysia

3Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

ABSTRACT The diversity and abundance of Dacinae fruit flies of the family Tephritidae were studied at

seven localities in the Pekan District, Pahang Darul Makmur, in the east coast of Peninsular Malaysia. Male

fruit flies were collected in the morning by using the attractants methyl eugenol and cue-lure. Three species

(Bactrocera carambolae, Bactrocera papayae and Bactrocera umbrosa) of the methyl eugenol group were

recorded. Bactrocera carambolae was present in all seven localities, being most abundant in a forest fringe

locality near a river but uncommon in the two Sungai Bebar localities. Bactrocera papayae was present in four

localities with human habitation but absent in the three forest areas. It was most common in the two localities

in Chini 2 with village setting. Bactrocera umbrosa was found in three localities with typical village setting with

fruit trees of Artocarpus species. The cue-lure group was present in five of the seven localities studied. Four

species were present – Bactrocera infesta, Bactrocera melastomatos, Bactrocera nigrotibialis and Bactrocera

cf tau. Bactrocera nigrotibialis was the predominant species while B. infesta occurred in quite large number

in a river bank locality. The other two species (B. melastomatos and B. cf tau) appeared to be uncommon.

Bactrocera infesta represented the first documented record for Peninsular Malaysia.

Keywords Bactrocera species – Bactrocera infesta – methyl eugenol – cue-lure – Pekan District Pahang –

new record

INTRODUCTION

Fruit flies of the family Tephritidae are plant feeders.

Many members of the subfamily Dacinae are of great

economic and agricultural importance because of

damage caused to commercial fruits and vegetables.

The damage, if uncontrolled, may result in a total loss

of the crop. The subfamily is represented by some 800

species worldwide, with some 300 species in Asia/

South-east Asia [1, 2].

Dacinae fruit flies of the genus Bactrocera were

previously referred to the genus Dacus [1]. They may

be polyphagous or exhibit great specificity for host

fruits [3]. The degree of host specificity appears to

be related to the extent of genetic diversity [4]. These

fruit flies also show variability in their attraction to

male chemical lures [3].

Most of the studies on Dacinae fruit flies concern

the species of agricultural importance. The present

paper reports the diversity and abundance of Dacinae

fruit flies, as determined by application of male

chemical lures, in seven localities – urban areas in

Chini 2, and settlement and forest areas in Runchang

and Sungai Bebar – within the Pekan District of

the State of Pahang Darul Makmur, Peninsular

Malaysia.

MATERIALS AND METHODS

During a field trip in September 2009, we studied

the diversity and abundance of Dacinae fruit flies

in seven localities at Chini 2, Runchang and Sungai

Bebar. These localities are situated in the Pekan

District, Pahang Darul Makmur, in the east coast of

Jostt vol 6.indd 17 7/22/10 10:08:41 PM

17


18

Journal of Science and Technology in the Tropics (2010) 6: 17-21

Peninsular Malaysia. Chini 2 is a small town of the

Chini Felda scheme while Runchang is an Orang Asli

settlement in the Sungai Bebar area. The surrounding

of Sungai Bebar is peat swamp forest.

Male fruit flies were collected in the morning by

means of the sex attractants methyl eugenol (4-allyl-1,

2-dimethoxybenzene) and cue-lure (4-[4-(acetyloxy)

phenyl]-2-butanone). A small amount of each lure

was applied separately and away from each other on

the upper surface of a green leaf – the lures are very

effective and not affected by the leaf. Insects attracted

to the lures over a period of about 30 minutes were

collected with the aid of specimen tubes and plastic

bags. The specimens were preserved in ethanol in

the field and subsequently identified using current

literature and personal experience [1, 2, 5, 6].

Representative individuals were also photographed

in the field.

The diversity of the fruit flies was analysed using

BioDiversity Professional Ver 2.0 1997. Shannon-

Weiner index and Jaccard cluster analysis were

applied for this study.

RESULTS

The diversity and relative abundance of Dacinae

fruit flies collected in Chini 2, Runchang and Sungai

Bebar are listed in Table 1. Three species of methyl

eugenol group were present – Bactrocera carambolae

Drew and Hancock, Bactrocera papayae Drew and

Hancock and Bactrocera umbrosa (Fabricius). The

cue-lure attractant yielded four species – Bactrocera

infesta (Enderlein), Bactrocera melastomatos Drew

and Hancock, Bactrocera nigrotibialis (Perkins) and

Bactrocera cf tau.

For the methyl eugenol group all three species were

present in three localities, C2, R1 and R2. Two species

(B. carambolae and B. papayae) were collected in

C1 and only B. carambolae was found in R3, SB1

and SB2. Fruit flies of the cue-lure group were found

in five of the seven localities studied, not present in

C2 and R2 (Table 1). Bactrocera nigrotibialis was

the predominant species while B. infesta occurred in

quite large number in a river bank locality. The other

two species (B. melastomatos and B. cf tau) appeared

to be uncommon.

The Shannon-Weiner diversity index of the

Dacinae fruit flies revealed that R1 had the highest

H’ (0.58) followed by R2 (H’= 0.432) and C2 (H’=

0.401) (Table 1). Locations SB1 and SB2 yielded the

lowest H’ of 0.126 and 0.110 respectively (Table 1).

The Jaccard cluster analysis showed that R2 and C2,

and SB2 and R3 had respectively similarity of 100%

(Fig. 1).

DISCUSSION

An extensive trapping effort, using Steiner traps

baited with methyl eugenol or cue-lure and provided

with malathion, was conducted over 12-36 months in

the 1990s in Peninsular Malaysia and Thailand [7].

The trapped fruit flies were collected on a 1-2 week

Table 1. Species and number of Dacinae fruit flies collected in September 2009 by methyl eugenol

and cue-lure attractants from Chini 2, Runchang and Sg. Bebar (Pekan District, Pahang, Peninsular

Malaysia). C1, edge of secondary vegetation and fence of apartments in Chini 2; C2, abandoned village

house along main road and near housing estate in Chini 2; R1, near school and Orang Asli house with

orchard and secondary vegetation in Runchang; R2, near Orang Asli house in Runchang; R3, secondary

forest near river in Runchang; SB1, river bank near abandoned aquaculture area along Sg. Bebar; SB2,

forested area upstream from SB1 of Sg. Bebar.

Lure/species C1 C2 R1 R2 R3 SB1 SB2

Methyl eugenol

B. carambolae

B. papayae

B. umbrosa

Cue-lure

B. infesta

B. melastomatos

B. nigrotibialis

B. cf tau

22

58

2

28

26

5

12

16

8

1

1

1

4

6

12

63 1 3

Number of species 3 3 6 3 2 3 2

Shannon H’ 0.299 0.401 0.580 0.432 0.298 0.126 0.110

Jostt vol 6.indd 18 7/22/10 10:08:42 PM

49

28

1

40


asis. There were 12 stations in the state of Pahang,

Peninsular Malaysia – the exact localities were however

not stated, but presumably in agricultural areas. In

that exercise, only three species (B. carambolae,

B. papayae and B. umbrosa) of the methyl eugenol

group and a single species (B. cucurbitae) of the cuelure

group were recorded in Pahang.

In another study carried out in the Endau-Rompin

rain forest in southern Peninsular Malaysia from

August to November 1985, tephritid fruit flies were

collected by ‘Malaise trap’, hand collection, and

methyl eugenol baited and cue-lure baited traps

[8]. The lure-baited traps were set for four days per

month, with one trap for methyl eugenol and four for

cue-lure. This study yielded four Bactrocera species

and 28 non-Dacinae species – quantitative data were

not provided. The methyl eugenol baited traps yielded

fruit flies of the B. dorsalis complex – B. dorsalis

(Hendel) does not occur in Peninsular Malaysia and

the complex consists of many species [5]. The cuelure

baited traps produced two species, B. nigrotibialis

and B. tau. Bactrocera latifrons (Hendel), which is

not attracted to both methyl eugenol and cue-lure,

was collected in the Malaise trap, in addition to B.

dorsalis complex and B. tau.

The diversity of Bactrocera species in the present

study is comparable to studies using lure-baited traps

(with male attractants methyl eugenol and cue-lure). It

is also similar to studies employing the same method of

applying the lures on green leaves [9, 10]. The study in

Negeri Sembilan yielded two species (B. carambolae

and B. papayae) of the methyl eugenol group and

three species (B. melastomatos, B. nigrotibialis and

Jaccard Cluster Analysis (Simple Average Link)

0. % Similarity

50.

SB1

SB2

R3

R1

R2

C2

C1

100

B. tau) of the cue-lure group [9]. In Kelantan, the

two localities studied (Pantai Melawi and Selising)

supported different species and different density of

the common species – B. carambolae, B. papayae,

B. melastomatos in both localities, B. caudata only

in Pantai Melawi, and B. umbrosa only in Selising

[10]. The presence or absence of Bactrocera species

and their abundance are dependent on the availability

of their host plants/fruits [11]. For example, in the

present study B. umbrosa was found only in localities

with typical village setting. This species is rather

host specific [12], infesting fruits of Artocarpus spp.

which were not evident in the localities where it was

not collected.

In the extensive study carried out in Pahang [7],

the Bactrocera species showed seasonal abundance

in number. Bactrocera carambolae, B. papayae and

B. umbrosa had higher number in September than

other months, while B. cucurbitae showed a peak in

November. The present study carried out in September

2009 did not yield B. cucurbitae; it may be attributed

to the absence of host plants Cucurbitaceae which

were not evident in all the seven localities studied.

Of the cue-lure group of fruit flies, B. nigrotibialis

(Fig. 2) was the predominant species. It occurred in

large number in two forest areas but only a single

individual was encountered in an inhabited area. It

was present in the Endau-Rompin rain forest [8] but

not recorded in the extensive trapping carried out in

12 stations in Pahang [7]. This species is not confined

to the forest as it is present in the University of Malaya

campus (unpublished data).

An unexpected find in the present study was the

large number of B. infesta (Fig. 3) attracted to cuelure

in locality SB1 (river bank near an abandoned

Figure 1. Dendrogram of Dacinae fruit flies species

similarity of different localities in Chini District,

Pekan, Pahang . Figure 2. Male Bactrocera nigrotibialis.

Jostt vol 6.indd 19 7/22/10 10:08:44 PM


19

Journal of Science and Technology in the Tropics (2010) 6: 17-21


20

Journal of Science and Technology in the Tropics (2010) 6: 17-21

aquaculture area along Sg. Bebar). As far as we

are aware there is no published record of this

species for Peninsular Malaysia – we had however

encountered it before but not published. It was

originally described as Polistomimetus infestus

Enderlein 1920 and later treated as Dacus

(Pacifodacus) infestus [7]. It looks remarkably like

Callantra sphaeroidalis (Bezzi) but the first two

segments of the antennae are short [6]. The typelocality

is Sumatra; it has been recorded in Indonesia,

Thailand and Laos. It is a rufous species with

only a short median yellow vitta, a petiolate abdomen,

a very large apical spot in the wing, and a complete

black band along lower surface of face [6]. As far

as known, there is no published information on its

attraction to lure and host plants.

The present method of using chemical attractants

does not uncover the presence of tephritid species

that do not respond to such lures [3], e.g. B. arecae

and B. latifrons. In the study using four collection

methods in the Endau-Rompin rain forest, Malaise

traps accounted for 20 out of 32 species and hand

collection 12 species, compared to three species by

methyl eugenol and cue-lure [8].

In sum, different localities in the present study

yielded different composition and abundance of

Bactrocera species, due most likely to the presence

and availability of host plants/fruits. Of the seven

species present, B. infesta constitutes a new record

for Malaysia. The higher number of Bactrocera

species and Shannon H’ in localities R1, R2 and C2

are attributed to the presence and availability of host/

fruit and orchards in or near the localities. These

areas also showed higher similarity percentage as

indicated by Jaccard cluster analysis.

Acknowledgement – The field work was supported by

the research grant UKM-GUP-ASPL-07-04-048.

We thank our institutions (University of Malaya and

Universiti Kebangsaan Malaysia) for facilities and

financial support; and the Forestry Department of

Pahang Darul Makmur for permission to carry out field

research.

Figure 3. Male Bactrocera infesta.

Jostt vol 6.indd 20 7/22/10 10:08:47 PM




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Yong H.S. (1994) Host fruit preferences in two

sympatric taxa of the Bactrocera dorsalis complex

(Insecta: Diptera: Tephritidae). In Yong H.S. and Khoo

S.G. (Eds.) Current research on tropical fruit flies and

their management pp. 1-8. The Working Group on

Malaysian Fruit Flies, Kuala Lumpur.

Yong H.S. (1988) Allozyme variation in the Artocarpus

fruit fly Dacus umbrosus (Insecta: Tephritidae) from

Peninsular Malaysia. Comparative Biochemistry and

Physiology 91B: 85-89.

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Journal of Science and Technology in the Tropics (2010) 6: 17-21


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Journal of Science and Technology in the Tropics (2010) 6: 23-30

The mammal fauna of Pulau Singa Besar, Langkawi, Kedah,

Peninsular Malaysia

Lim Boo Liat1 , Norhayati Ahmad2,3 , O. Noor Alif Wira4 , Shahrul Anuar Md. Sah5 1Jalan Koop Cuepacs 3 E, Taman Cuepacs, 43200 Cheras, Selangor, Malaysia

2School of Environment and Natural Resource Sciences, Faculty of Science and Technology,

University Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

3Institute for Environment & Development (LESTARI) , Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor, Malaysia

4Department of Wildlife and National Parks, Penang, Malaysia

5Department of Biological Science, Universiti Sains Malaysia, Penang, Malaysia

(Email: chizlim54@yahoo.com)

Abstract Studies on the mammal fauna of Pulau Singa Besar, Langkawi were carried out during 2001

and 2002. A total of 51 species – 21 non volant and 30 volant species – from past and recent studies were

recorded. Two species, the Smooth otter (Lutrogale perspicillata) and Indian False vampire (Megaderma

lyra) are new records for the Langkawi Archipelago. The species diversity between the non volant and volant

mammals is discussed.

Keywords mammals – Malaysia – bats – diversity – island

INTRODUCTION

Pulau Singar Besar, Langkawi is one of the 104 islands

in the Langkawi Island archipelago. It is a permanent

forest reserve, uninhabited, and was established as a

bird and animal sanctuary in 1990. The island consists

of various pristine forest types, such as dipterocarp

lowland tropical forest, mangrove coastal forest, and

sandy beach forest. In terms of the mammal fauna,

the island has been known for the high density of

mousedeer (Tragulus kanchil, T. napu), wildboar

(Sus scrofa), and Long-tailed macaque (Macaca

fascicularis).

Not much was known of its mammal species

diversity until the late 1980s. A survey was carried out

on the vertebrate fauna and 17 species of mammals

were recorded [1]. After a lapse of 10 years, more

comprehensive studies on the vertebrate fauna

(amphibian and reptile, mammal, bird) were carried

out at different periods of 8–10 days each in 2001

and 2002 under the purview of the Department of

Wildlife and National Parks (DWNP). We report here

the results of the 47 mammal species.

MATERIALS AND METHODS

Pulau Singa Besar is located south of the main

Langkawi island, wedged in between the small island

of Pulau Beras Basah on the west and the larger

island of Pulau Dayang Bunting on the east (Fig.

1). The island is 11.3 km 2 in area, reaches 270 m in

elevation and is nearly entirely covered by pristine

dipterocarp forest. The island is traversed by steep

valleys drained by seasonal streams. Limestone

outcrops are prominent on the north-eastern part of

the island. Caves of varying depths and widths occur

in this limestone area. There are three clear water

streams that flow to the east of the island, Sg. Boton

and Sg. Pantai, while Sg. Sepai flows to the northern

coast.

The island, as in the Kadawi region, experiences

dry periods between December-April. During the

north-east monsoon, the island is exposed to torrential

rain and storms. Day temperature varies from 27°C

under shade and 37ºC in the open on the beach. Night

temperature is around 24–27°C. It is hot and humid

most of the time [1].

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Journal of Science and Technology in the Tropics (2009) 5: 111-116


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Journal of Science and Technology in the Tropics (2010) 6: 23-30

Figure 1. Map of Pulau Singa Besar and the trails where trapping of mammals was carried out.

Jostt vol 6.indd 24 7/22/10 10:09:02 PM


The field team comprised eight members, six of

whom were from the DWNP, Kuala Lumpur. Three

field trips were carried out: 2-14 May 2001, 2-8

February 2002, and 2-13 June 2002. Trapping of

the mammals was carried out at designated trails:

Mangrove boardwalk (MBW), Cicada trail (CT),

Perdana trail (PT), Dusun trail (DT), Sungai Sepai

trail (SS) and the Staff quarters (SQ). Trappings were

carried out by four field workers along two trails for

each of the field trip. Fifty collapsible traps and two

harp traps were deployed at each sampling site. Thus,

the three field trips covered all the six trapping sites. All

mammals captured were processed, i.e. measurements

were taken and reproductive conditions noted. While

visiting the various traps during the day and night, any

nocturnal and diurnal animals sighted were noted and

identified up to the species level whenever possible.

Voucher specimens for some of the volant mammals

(1–4 individuals) were collected and preserved using

ethanol and some of the non-volant species were

skinned for voucher specimens. All the voucher

specimens were deposited in the museum collection at

the Institute of Biodiversity, Bukit Rengit, Lanchang,

Pahang, Peninsular Malaysia.

Mangrove boardwalk (MB)

The mangrove boardwalk meanders through a patch

of mangrove forest next to the Staff quarters (SQ)

and Interpretation Center (IC) building just before

entering the forest. Rat traps were laid throughout the

200-m length of the boardwalk. Harp traps were set at

2 m apart at the mid-point on the boardwalk.

Cicada trail (CT)

This trail starts from a small section of the boardwalk

proceeding along the sandy beach. It is about 2 km

long. The trail is flanked on the left by disturbed

forest and the right by the sea coast. It is a popular trail

among tourists during weekends and public holidays.

Traps were laid in the forest and at the fringe. Harp

traps were set in the forest.

Perdana trail (PT)

The trail starts from the end of CT, heads south-west

through various forest types (scrub, secondary and

primary forests), meanders to northeast and ends at

Sg. Sepai Valley (SS). The terrain is undulating with

some parts very steep. The trail is about 6 km from

PT to SSV. As the trail is very long, the 50 traps were

divided into two areas along the trail at 25 m interval.

The first 25 traps were spread out along 1.5 km from

the beginning of the CT and ended at the base of the

steep point at about 800 m high; the second set of 25

traps were laid just below the steep base and ended at

the SSV. The harp traps were set at the midpoint of PT

and the other at the midpoint of SSV.

Dusun trail (DT)

The trail starts at 1.5 km of the PT, takes a 45 degree

turn to the right and descends another 0.5 km to a

small orchard of mostly durian trees. Another way of

getting to the DT is via boat during high tide through

the mangrove forest. Traps were laid in the plantations,

fringe of mangrove and along forest trails and stream

banks. Harp traps were set in the plantations and also

in the entrance of crevices of rock boulders in the

area.

Sungai Sepai valley (SS)

This area is one of the campsites for visitors to the

island. The forest here is disturbed primary forest

edged by a small stretch of mangrove forest. Deeper

into the secondary forest is pristine forest habitat. A

large forest stream, about 1-2 m wide undulating to

a stretch of limestone rocks, provides the source of

clean water for the campsite. Traps and harp traps

were set in the primary part of the forest.

Staff quarters (SQ)

There are four buildings making up the staff quarters

next to the MW and IC near the jetty. Traps and harp

traps were set at a stretch of disturbed forest behind

the SQ.

RESULTS AND DISCUSSION

A total of 47 mammals was recorded from the six

study sites from the three field trips in 2001 and 2002

on the island. This comprised 18 species of nonvolant

mammals from 12 families and 29 species of

volant mammals from eight families (Appendix 1).

The greater number of volant species was probably

due to the availability of roosting sites (crevices of

rock boulders, caves, hollow trees) in the island. The

low species diversity of the non volant mammals in

island ecosystem is generally low and Pulau Singa

Besar is no exception. This is also shown on studies

of other islands, for example, 45 mammal species

(including 19 bat species) in Pulau Tioman, Pahang

and 23 species (including 15 bat species ) in Pulau

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Journal of Science and Technology in the Tropics (2010) 6: 23-30


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Journal of Science and Technology in the Tropics (2010) 6: 23-30

Perhentian [2-4]. This is due to the isolation from

the mainland where emigration and immigration are

marginal and also limited sizes of the landmass. In

the case of arboreal species (bats and birds), they

are very mobile and if it is within their flight range,

the island would serve as part of their transit shelters

for resident and migrant animals, as well as for food

resources.

Among the six study sites, higher species richness,

ranging from 21–38 species were recorded at CT,

PT, DT, and SSV. The higher number of species is

associated with the diversity of habitats (scrub,

secondary and primary forests) found in each of these

study sites. The markedly lower species richness

recorded at MBW (8 species) and SQ (12 species)

was probably due to the less diverse habitat of the

coastal mangrove forest as compared to the other

sites (Appendix 1).

Non-volant mammals

All the 18 mammal species recorded in the island

(Appendix 1: No. 1-18) are common species, except

the Smooth otter (Lutrogale perspicillata), which is a

new record for Langkawi. Most abundant among the

18 species were the tree-shrew, long-tailed macaque,

plantain squirrel, three species of field and forest rats,

wild boar and mouse-deer (Appendix 1: Nos. 4, 5,

7, 9-11, 17, and 18). The least encountered was the

White-toothed ground shrew (Crocidura fuliginosa)

(Appendix 1: No 1). This species is a tiny, burrowing

animal. Pit fall traps have been proven to be the most

suitable trapping technique to obtain this species [5].

Previous study [1] recorded 15 species of nonvolant

mammals on Pulau Singa Besar, of which, four

species were additional records for the island: the Slow

loris (Nycticebus coucang), Black giant tree squirrel

(Ratufa bicolor), Eurasian otter (Lutra lutra) and the

Three-striped palm civet (Arctogalidia trivirgata).

With these, the non-volant mammals increased to 22

species on Pulau Singa Besar. Of these, 20 species

were recorded on the main Langkawi island [6],

except Niviventer cremoriventer and Lutrogale

perspicillata. However, all the 22 species recorded

from Pulau Singa Besar are common in the mainland

of Peninsular Malaysia, except the Eurasian otter.

Volant mammals

The 29 species of bats recorded on the island comprised

seven species of fruit bats (Pteropodidae) and 22

species of insectivorous bats from seven families:

Emballonuridae, Nycteridae, Megadermatidae,

Rhinolophidae, Hipposiderosidae, Vespertilionidae

and Molossidae (Appendix 1: No. 19–47).

Fruit bats

All the seven species of fruit bats are common on the

island; the Lesser Dog-faced fruit bat (Cynopterus

brachyotis) and the Cave fruit bat (Cynopterus

Eonycteris spelaea) (Appendix 1; No. 20, 22) were

more common than the rest (Appendix 1: No. 19,

21, 23-25). Both these species were netted in all the

six study sites with greater frequency of captures

(27, 17 individuals respectively) compared to the

others which were netted between 2-4 study sites and

with less frequency of captures, ranging from 4–12

individuals.

Mustaffa Babjee [1] recorded four species of fruit

bats on the island. One of them, the Island Flying fox

(Pteropus hypomelanus) was a new locality record

for Langkawi, which increased the fruit bat fauna to

eight species from the island to date, compared to five

species in the main Langkawi island [6]. However,

all the eight species recorded on the island are also

common in lowland and hill forests in mainland

Peninsula Malaysia.

Insectivorous bats

The species richness of this group was reflected by the

troglodytic habits where the roosting environments

are available on the island. At least 12 species

(Appendix 1: No. 29-33, 36-40,42, 45) were cave

dwellers [7-9]. Among the 22 species, three species,

the Intermdiate Horseshose bat (Rhinolophus affinis),

Bicolored Roundleaf bat (Hipposideros bicolor) and

Common Roundleaf bat (Hipposideros galeritus),

were common with high frequency of captures (44,

38, and 27, respectively). Of particular interest was a

species with a single specimen netted, the Indian False

vampire (Megaderma lyra), which is uncommon.

This species is carnivorous in habit [6].

Fourteen of the 22 species on the island were also

recorded on the main Langkawi island [6]. On the

mainland of Peninsular Malaysia, all the 22 species

are common in lowland and hill forests.

Reproductive status

The examination of pregnant females was based

on visible observation of advance pregnancy of

the animals. This method was used to conserve the

species from being sacrificed for detail examination

Jostt vol 6.indd 26 7/22/10 10:09:03 PM


of the reproductive tracts, and also to prevent the

animal from being overly stressed by holding it too

long before release.

A total of 35 species, comprising eight species of

non-volant (63 individuals) and 27 species of volant

mammals (138 individuals), was examined in May

2001 and February and June 2002 (Table 1). Of the

non-volant mammals, two species (9 individuals)

were not gravid. For the females of the other six

species (Table 1: No. 1-2, 4-6, 8) with 54 individuals,

about 33% (18 individuals) were visibly pregnant.

The number of individuals pregnant during the three

periods showed no marked difference, being 5, 7, and

6 respectively.

Of the 27 species of volant mammals examined,

13 species (26 individuals) were not gravid. In the

other 14 species (Table 1: No. 9-10, 12-14, 19, 21,

24-25, 28, 30, 33-35) with 112 individuals, 36.7%

(41 individuals) were visibly pregnant. The number

of individuals pregnant during May 2001, February

and June 2002 was 15, 4, and 22 which suggests that

there was a high birth trend in May and June.

Table 1: Number of visibly pregnant females of non-volnt and volant mammals of Pulau Singa Besar, Langkawi,

Kedah, Peninsular Malaysia.

No. Species

No. Indiv.

Examined

2-14 May

2001

2-12 Feb

2002

2-13 Jun

2002

NON-VOLANT MAMMALS

1 Tupaia glis 7 2 2

2 Callosciurus notatus 7 1 1

3 C. caniceps 5

4 Rattus tiomanicus viclana 21 4 3 5 12

5 Rattus exulans 7 1 1

6 Leopoldamys sabanus 8 1 1

7 Niviventer crimoriventer 4

8 Maxomys surifer 4 1 1

VOLANT MAMMALS

9 Rousettus amplexicaudatus 2 1 1

10 Cynopterus brachyotis 12 1 3 4

11 C. horsfieldii 2

12 Eonycteris speleae 9 1 2 3

13 Penthetor lucasi 8 1 1

14 Macroglossus minimus 5 1 1

15 Emballonura monticola 4

16 Taphozous melanopogon 1

17 Nycteris tragata 4

18 Megaderma spasma 1

19 Rhinolophus affinis 24 4 2 5 11

20 R. trifoliatus 3

21 R. coelophyllus 3 1 1

22 R. pusilllus 2

23 R. lepidus 3

24 Hipposideros bicolor 21 4 1 6 11

25 H. galeritus 11 1 2 3

26 H. cinareus 1

27 H. diadema 2

28 H. larvatus 5 1 1

29 Myotis muricola 1

30 M. hasseltii 1 1 1

31 Murina suillus 1

32 Kerivoula papillosa 1

33 K. intermedia 4 1 1

34 Scotophilus kuhli 4 1 1

35 Tadarida mops 3 1 1

Total 201 20 11 28 59

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Total

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Journal of Science and Technology in the Tropics (2010) 6: 23-30


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Journal of Science and Technology in the Tropics (2010) 6: 23-30

Conclusion

The high species richness of the chiropteran

fauna in Pulau Singa Besar could be due to: (1)

the island with its pristine forest and surrounded

by limestone outcrops and traversed by valleys

with seasonal streams, provides abundant

roosting sites, such as caves, crevices of rock

boulders, tree holes, etc.; and (2) bats could fly

from neighbouring Pulau Basah on the west and

the larger Pulau Dayang Bunting on the east.

The non volant mammals are mostly terrestrial

residential species where emigration-immigration

is limited. Thus, the low species richness of this

1. Mustaffa Babjee A. (1990) Pulau Singa Langkawi:

bird and animal sanctuary. Department of Veterinary

Sciences, Ministry of Agriculture, Malaysia.

2.

3.

4.

5.

Lim B.L., Kelvin K.P.L. and Yong H.S. (1999) The

terrestrial mammals of Pulau Tioman, Peninsular

Malaysia, with a catalogue of specimens at the Raffles

Museum, National University of Singapore. Raffles

Bulletin of Zoology 6: 101-123.

Yong H.S. (1974) Diversity and abundance of small

mammals in two Malaysian offshore islands. Malayan

Nature Journal 27: 27-29.

Tamblyn A., Malley R., Turner C. and Hughes T.

(2009) The bat fauna (Mammalia, Chiroptera) of

Pulau Perhentian, Peninsular Malaysia. Malayan

Nature Journal 61(1): 10-22.

Nor Zalipah M., Juliana S. and Lim B.L. (2007) A note

REFERENCES

group of animals in the island ecosystem is a

natural phenomenon [10].

With the additional four species, Pteropus

hypomelanus, Nycticebus coucang, Lutra lutra

and Ratufa bicolor recorded in earlier study [1],

the mammal fauna of the island now stands at 51

species.

Acknowledgements – We are grateful to the staff of

DWNP, particularly the Director-General, Dato’ Musa

Nordin (retired) for his support; and the General Manager

of the Langkawi Development Authority (LADA), for

supporting the logistic in the field.

on the arboreal monkey-footed rat in a pit-fall trap.

Journal of Wildlife and Parks 24: 63-65.

6. Medway Lord (1983) The wild mammals of Malaya

(Peninsular Malaysia) and Singapore. 3rd Edn. Oxford

University Press, Kuala Lumpur.

7. Medway, Lord (1965) Identification of Malaysian

cave bats. Malayan Nature Journal 19: 88-107.

8. Lim B.L. (1967) Abundance and distribution of

Malaysian bats in different ecological habitats. Federal

Museum Journal (N.S.) II (1966): 61-76.

9. Norhayati A., Shukor M.N., Shahrul Anuar M.S.,

Nurul Ain E. and Norzalipah M. (2007) Note on the

bats of Gua Kelawar, Langkawi Island, Kedah. Journal

of Wildlife and Parks 24: 1-6.

10. Alle W.C. and Schmidt X. (1951) Ecological animal

geography. Chapman and Hall Ltd., London.

Jostt vol 6.indd 28 7/22/10 10:09:05 PM


Appendix 1: Mammal species recorded on Pulau Singa Besar, Langkawi, Kedah, Peninsular Malaysia. (MB=Mangrove

Boardwalk, CT=Cicada Trail, PT=Perdana Trail, DT=Dusun Trail, SS=Sungai Sepai Valley, SQ=Staff Quarters;

S=Sighted, S-TR=Sighted and track signs, R=Released).

No. Order/Family/Species

INSECTIVORA

Soricidae

MB CT PT DT SS SQ

Total

Male Female

Voucher

No.

1 Crocidura fuliginosa 1 1 0 35

DERMOPTERA

Galeopterida

2 Galeopterus variegatus S - 1 1 -

PHOLIDOTA

Manidae

3 Manis javanicus S -1 1 -

SCANDENTIA

Tupaiidae

4 Tupaia glis 2 1 1 4 3 2 6 7 36-37

PRIMATE

Cercopithecidae

5 Macaca fascicularis S S S S S S - - -

6 Trachypithecus obscurus S S - - -

RODENTIA

Sciuridae

7 Callosciurus notatus S - 1 1 2 1 1 4 3 7 38-40

8 C. caniceps 1 2 1 4 1 4 5 41

Muridae

9 Rattus tiomanicus viclana 12 12 5 2 6 6 21 42-43

10 Rattus exulans 2 6 2 3 2 8 7 44-45

11 Leopoldamys sabanus 4 6 2 3 7 8 46-47

12 Niviventer cremoriventer 3 2 1 4 48-49

13 Maxomys surifer 1 1 2 4 4 4 51-

Hystricidae

14 Hystrix brachyura S - 1 S - 1 2 -

CARNIVORA

Viveridae

15 Paradoxurus hermaphroditus S - 1 S - 2 S - 1 1 S - 1 1 2 5 R

Mustelidae

16 Lutragale perspicillata S - 2 S - 2 4 -

ARCTIODACTYLA

Suidae

17 Sus scrofa S S S + Tr S + Tr S - 3 4 -

Tragulidae

18 Tragulus kancil/napu S - 2 S - 1 S - 1 S - 2 6 -

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Journal of Science and Technology in the Tropics (2010) 6: 23-30


30

Journal of Science and Technology in the Tropics (2010) 6: 23-30

CHIROPTERA

Pteropodidae

19 Rousettus amplexicaudatus 1 4 3 2 52

20 Cynopterus brachyotis 2 5 3 8 5 3 14 12 53-54

21 C. horsfieldii 1 3 2 4 2 55

22 Eonycteris spelaea 1 1 3 6 4 2 8 9 56-57

23 Penthetor lucasi 2 4 4 2 4 8 58

24 Macroglossus minimus 2 2 4 2 5 5 59

25 Balionycteris maculata 2 2 3 1 60

Emballonuridae

26 Emballonura monticola 1 3 3 3 4 61

27 Taphozous melanopogon 1 2 2 1 62

Nycteridae

28 Nycteris tragata 2 2 2 2 4 63

Megadermatidae

29 Megaderma spasma 3 2 1 64

30 M. lyra 1 1 65

Rhinolophoridae

31 Rhinolophus affinis 12 8 13 11 20 24 66-67

32 R. trifoliatus 3 2 2 3 68-69

33 R. coelophyllus 3 2 3 5 3 70-71

34 R. pusillus 3 1 2 2 72-73

35 R. lepidus 1 3 2 3 3 74-75

Hipposideridae

36 Hipposideros bicolor 11 12 9 6 17 21 76-78

37 H. galeritus 3 10 14 16 11 79-80

38 H. cineraceus 2 1 1 81

39 H. diadema 3 1 2 82

40 H. larvatus 2 5 2 4 5 83-84

Vespertilionidae

41 Myotis muricolor 3 2 1 85

42 M. hasseltii 1 3 3 1 86-87

43 Murina suillus 1 2 1 3 1 88-89

44 Kerivoula papillosa 3 2 1 90

45 K. intermedia 2 4 2 4 93

46 Scotophilus kuhli 2 4 2 4 94

Molossidae

47 Tadarida mops 2 3 2 3 95-96

Total no. species 8 21 28 36 38 12

Total No. Individuals 12 64 86 101 112 30

Jostt vol 6.indd 30 7/22/10 10:09:07 PM


Journal of Science and Technology in the Tropics (2010) 6: 31-38

Antiproliferative and antioxidant properties of leaf extracts of Pereskia bleo

(Cactaceae) and their ability to limit natural and oxidant-induced

apoptotic cell death

Hui Meng Er * , Hong Lim Lee, Ammu Kutty Radhakrishnan

Faculty of Medicine and Health, International Medical University, No.126 Jalan 19/155B, Bukit Jalil,

57000 Kuala Lumpur, Malaysia

( * Corresponding author E-mail: huimeng_er@imu.edu.my)

Abstract The anti-proliferative activities of methanol extract of leaves of Pereskia bleo and its ethyl acetate,

t-butanol and aqueous fractions were studied in normal mouse fibroblast cells (NIH/3T3) and mouse

mammary cancer cells (4T1). Although the extracts did not show significant anti-proliferative effect, they

exhibited selectivity in inhibiting the proliferation of the cancer cells (4T1) compared to the normal cells (3T3).

The antioxidant properties of the leaf extracts and their ability to limit natural and oxidant-induced cell death

were also studied. Among the extracts, the t-butanol extract possessed the highest antioxidant property. The

extracts were also able to limit natural and oxidant-induced cell death in normal mouse fibroblast cells.

Keywords Pereskia bleo leaf extracts – anti-proliferative – antioxidant – apoptotic cell death

INTRODUCTION

Pereskia bleo (Kunth) DC (Cactaceae) is commonly

consumed by some ethnic groups in Malaysia for its

medicinal properties. It is claimed to have antidiabetic

and antihypertensive properties [1]. Besides, it is

widely believed by the local community that the

drink prepared by boiling the plant leaves in water is

effective in preventing and treating cancers. Hence,

the plant is commonly cultivated in the gardens.

However, there are very little in vitro and in vivo

studies to support most of these claims.

It has been shown previously that the methanol

extract of P. bleo can kill T47-D human mammary

cancer cells [2], suggesting that this plant has the

potential to be developed as a candidate for treatment

of cancers. However, in another study, it was reported

that the methanol and aqueous extracts of the leaves

did not possess any significant anti-proliferative

activity on mouse mammary cancer cells (4T1) and

normal mouse fibroblast cells (3T3) [3]. Moreover,

it was also reported that the aqueous extracts of the

leaves could form mutagenic compounds when these

are metabolized by liver enzymes [3].

Although the methanol extract and its ethyl-acetate

fraction has been reported to have high cytotoxic effect

on human nasopharyngeal epidermoid carcinoma

(KB) cells [4], their cytotoxic activity in other cell

lines such as human cervical carcinoma cell line

(CasKi), human colon carcinoma cell line (HCT116),

hormone-dependent breast carcionoma cell line

(MCF-7), and non-cancer human fibroblast cell line

(MRC-5) was less significant [4]. In another study

it was reported that the hexane, dichroloromethane,

ethyl acetate and methanol extracts of the leaves were

also non-effective in inducing cell death towards

MCF-7 (human breast cancer), HT-29 (human colon

carcinoma) and CEM-SS (human T4-lymphoblastoid)

cell lines [5].

Anti-proliferative studies carried out on the

methanol extract of the stem and its ethyl acetate,

t-butanol and aqueous fractions on mouse mammary

cancer cells (4T1) and normal mouse fibroblast cells

(NIH/3T3) did not indicate significant antiproliferative

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Journal of Science and Technology in the Tropics (2009) 5: 117-123


32

Journal of Science and Technology in the Tropics (2010) 6: 31-38

activity [6]. A detailed analysis on the methodologies

employed in the previous studies suggested that

the inconsistent findings on the anti-proliferative

activity could be attributed to differences in the parts

of the plant studied (e.g. leaf, stem, whole plant),

plant extraction procedure, cell lines used and antiproliferation

assay conditions [3].

The diversity of secondary metabolites in plants

is well known. They play a major role in contributing

to the plants’ natural defense mechanism against

predators or diseases caused by microorganisms. As

the stems of P. bleo are heavily covered with sharp

spines that can protect the plant against preys, they

may also be associated with high level of secondary

metabolites of important medicinal properties, such

as anticancer activity. However, previous study has

shown that the stem extracts do not possess significant

anti-proliferative activity against NIH/3T3 and 4T1

cells [6]. In the current study, the same procedure

was employed for the extraction and fractionation of

the leaves of the plant using the same solvents, and

the anti-proliferative activities of the extracts were

assessed using the same cell lines.

The mechanism of cell death caused by oxidative

stress has been studied widely [7]. The generation of

endogenous reactive oxygen or nitrogen species as

a result of oxidative stress can lead to tissue injury

implicated in many diseases. Many antioxidant

compounds are found in higher plants and they have

been shown to possess the ability to quench free

radicals and reactive oxygen species in vitro [8]. In

this study, the in vitro antioxidant property of the leaf

extracts, as well as their ability to limit natural and

oxidant-induced cell death were evaluated.

MATERIALS AND METHODS

Plant Material

The plant cuttings of P. bleo were collected from Taman

Pertanian (Agricultural Garden) of the Universiti

Putra Malaysia (UPM), Selangor, Malaysia. The

voucher specimen (No. ACP 0116) was deposited in

the herbarium of Institut Biosains of UPM.

Preparation of leaf extracts

The leaves of P. bleo were separated, cleaned with

water and dried in a convection oven at 40 o C until

consistent weights were obtained. The dried leaves

were blended into powder form using an electric

blender. A weighed amount (20 g) of the leaf powder

was extracted with methanol (250 mL) using a soxhlet

extractor for four hours. The process was repeated

five times in order to obtain sufficient amount of

the extracts for further testing and the extracts were

combined. The methanol solvent in the combined

extract was removed under reduced pressure at

40 o C using a rotary evaporator and the extract was

further dried using a vacuum concentrator at 40 o C

until a consistent weight was obtained. A portion of

the methanol extract was fractionated by partitioning

into ethyl acetate, t-butanol and water. The solvents

in these fractionated extracts were removed under

reduced pressure at 40 o C using a rotary evaporator.

The ethyl acetate and t-butanol extracts were further

dried to consistent weight using a vacuum concentrator

at 40 o C. The aqueous extract was freeze-dried to a

consistent weight. The yields of the methanol, ethyl

acetate, t-butanol and aqueous extracts were 7.7%,

0.2%, 3.8% and 7.1%, respectively, based on the dry

weight of the leaves.

ABTS radical cation scavenging activity of

P. bleo leaf extracts

The assay is based on the ability of substances with

antioxidant properties to scavenge 2,2’-azinobis-

(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)

radical cations, which are blue-green in colour and

absorb at 734 nm [9]. Any positive reaction will lead

to the decolourisation of the ABTS radical cations.

The methanol extracts of the leaves were dissolved

in 0.01 M phosphate buffered saline (PBS) while the

ethyl acetate and t-butanol extracts were dissolved

in 0.8% (v/v) DMSO. Trolox and vitamin C were

used as positive controls. To perform this assay, the

ABTS solution (715 µL, 5 mM) was mixed with

potassium persulphate solution (285 µL, 2.5 mM)

and the reaction mixture was kept in the dark at room

temperature for 12-16 hours for the generation of

ABTS radical cations.

The ABTS radical cation solution was

appropriately diluted with 0.01 M PBS to give an

absorbance of 0.7 ± 0.2 at 734 nm. To 990 µL of this

diluted ABTS radical cation solution, 10 µL of each

of the concentrations of the leaf extracts, Trolox or

vitamin C was added. The absorbance of the mixture

at 734 nm was recorded at every minute for a period

of 12 minutes. The experiment was repeated five

times for each concentration of the extracts (2.5

to 50 µg/mL for t-butanol extract; 5 to 100 µg/mL

for the other extracts), Trolox (0.25 to 2.5 mM) or

Jostt vol 6.indd 32 7/22/10 10:09:08 PM


vitamin C (0.25 to 2.5 mM). Lower concentrations

of the t-butanol extract were used compared to the

other extracts due to the difficulty in dissolving this

extract. The percentage inhibition of ABTS radical

cation at each time point was calculated using the

formula: Percentage inhibition = [(Initial absorbance

- Absorbance at time of interest) / Initial absorbance]

x 100%.

Cell lines

The NIH/3T3 (normal mouse fibroblast cell line)

and 4T1 (mouse mammary cancer cell line) cells

were purchased from the American Type Culture

Collection (ATCC, Rockville). They were cultured

in RPMI 1640 medium containing L-glutamine and

supplemented with 10% FBS, 1% HEPES buffer

solution, 1% sodium pyruvate (100 mM) and 0.5%

penicillin-streptomycin in a humidified 5% CO 2

incubator at 37 o C.

Anti-proliferation assay

Cell proliferation was analysed using the MTT

assay [10]. This assay is based on the ability

of the mitochondria of living cells to reduce

a chemical, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium

bromide (MTT), which is a

yellow tetrazolium salt, to purple formazan product

that is insoluble in the aqueous phase. The NIH/3T3

or 4T1 cells (100 µL of 5x10 4 cells) were cultured in

a sterile 96-well flat-bottom tissue culture plate. The

plate was incubated at 37 o C in a humidified 5% CO 2

incubator for two hours. The methanol, ethyl acetate,

t-butanol and aqueous extracts of the leaves were

appropriately diluted (50 to 800 µg/mL) in medium

containing 0.8% (v/v) dimethyl sulphoxide (DMSO).

Various concentrations (25 to 100 µg/mL) of cisplatin,

which was used as a positive control was also prepared

in culture medium containing 0.8% (v/v) DMSO. The

diluted solutions were filtered using sterile 0.22 µm

filter units. Following the two hour incubation, 100

µL of the diluted leaf extracts or cisplatin were added

to the 96-well culture plates containing the NIH/3T3

or 4T1 cells (in triplicates). The final concentrations

of the leaf extracts in this assay ranged from 12.5 to

400 µg/mL while cisplatin ranged from 1.6 to 50 µg/

mL. Cells cultured in medium containing 0.4 % (v/v)

DMSO were used as negative control. The plates

were incubated at 37 o C in a humidified CO 2 incubator

for 72 hours. Then MTT solution (20 µL, 5 mg/mL in

PBS) was added to each of the wells and the plate was

returned to the incubator for 4 hours. Following this,

the supernatant in each well was removed carefully

(the purple formazan product was attached to the

bottom of the well). Dimethylsulphoxide (50 µL)

was then added to each well to dissolve the purple

formazan product. The absorbance of the solution

in each well was determined using an ELISA plate

reader at 570 nm. The percentage cell viability in each

well was calculated by the formula: Percentage cell

viability = (Absorbance at 570 nm of treated well /

Absorbance at 570 nm of negative control) × 100%.

Cell death detection by enzyme-linked

immunosorbent assay (ELISA)

The NIH/3T3 cells were diluted in culture medium

to a concentration of 1 x 10 5 cells/mL. This diluted

cell suspension (50 µL) was added to the wells of a

sterile 96-well flat-bottom tissue culture plate. The

plate was left in a humidified 5% CO 2 incubator at

37 o C for two hours. The extracts from the leaves

(methanol, ethyl acetate, t-butanol and aqueous

extracts) were dissolved in DMSO and diluted with

the culture medium to a concentration of 100 and 200

µg/mL. The concentration of DMSO in the diluted

solutions was 0.8% (v/v). These solutions were

filtered using a sterile 0.22 µm filter unit. A 50 µL

aliquot of the diluted solution (200 or 100 µg/mL)

of the leaf extracts, Trolox or vitamin C was then

added (in triplicates) to the respective wells in the

plate containing NIH/3T3 cells at the concentration

of 5 x 10 3 cells per well. To test for the ability of

these extracts to protect NIH/3T3 cells against DPPH

(2,2-diphenyl-1-Picrylhydrazyl) induced cell death,

50 µL of 30 µM DPPH solution was added to the

relevant wells one hour after the NIH/3T3 cells were

incubated in the presence of the appropriately diluted

extracts, Trolox, Vitamin C or cultured medium. The

plate was incubated at 37 o C for 24 hours. Trolox and

vitamin C were used as the positive controls while

medium containing 0.4% (v/v) DMSO served as the

negative control.

Cell death due to apoptosis was performed using

the Cell Death Detection ELISA PLUS kit according to

the manufacturer’s instruction as described previously

[3]. This assay is based on a quantitative sandwichenzyme-immunoassay

principle that utilises mouse

monoclonal antibodies directed against DNA and

histones associated DNA fragments. Briefly, the plate

was centrifuged at 200 × g for 10 minutes at room

temperature after 24 hours. The supernatant was

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Journal of Science and Technology in the Tropics (2010) 6: 31-38


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Journal of Science and Technology in the Tropics (2010) 6: 31-38

completely removed. Then, the pellet in each well

was resuspended in 200 µL of lysis buffer (provided

by manufacturer) and the plate was incubated at

room temperature for 30 minutes. Following this,

the plate was centrifuged at 200 × g for 5 minutes.

An aliquot of 20 µL of the supernatant containing

the cytoplasmic fraction in each well was carefully

transferred into appropriate wells of a streptavidincoated

plate (provided by manufacturer) for further

analysis. Then, 80 µL of an immunoreagent (provided

by manufacturer) was added into each well. The plate

was covered with an adhesive foil and gently shaken

at 200 rpm for two hours. The solution in each well

was then removed and the wells were thoroughly

rinsed thrice with 300 µL of incubation buffer

(provided by manufacturer). Following this, 100 µL

of ABTS solution (provided by manufacturer) was

added into each well and the plate was gently shaken

at 200 rpm until suitable colour development was

obtained (20-30 minutes). The absorbance of each

well was determined using an ELISA plate reader at

405 nm with a reference wavelength of 490 nm. The

enrichment factor was calculated using the following

formula provided by the manufacturer: Enrichment

factor = (Absorbance at 405 nm of treated cells) /

(Absorbance at 405 nm of untreated cells).

RESULTS

ABTS radical cation scavenging activity of

P. bleo leaf extracts

The percentage inhibition of the ABTS radical

cations over 12 minutes by the leaf extracts at the

highest concentrations is shown in Figure 1. Figure

2 shows the percentage inhibition of ABTS radical

cations at the sixth minute versus concentrations

of the leaf extracts, as well as the linear regression

equations for the graphs of the leaf extracts. The

Trolox Equivalent (TE) per gram dry extract for the

leaf extracts are presented in Table 1. Among the

leaf extracts, the t-butanol leaf extract exhibited the

Table 1. Trolox Equivalent (TE) µmole

per gram dry extract of Pereskia bleo leaf

extract and vitamin C.

Extract TE ± S.D.

Methanol leaf extract 176 ± 2

Ethyl acetate leaf extract 208 ± 3

t-Butanol leaf extract 416 ± 15

Aqueous leaf extract 181 ± 2

Vitamin C 5158 ± 3

highest ABTS radical cation scavenging activity and

this was followed by the ethyl acetate leaf extract.

The methanol leaf extract and aqueous leaf extract

showed comparable ABTS radical cation scavenging

activity and were the lowest among the leaf extracts.

Anti-proliferation assay

There was a gradual decrease in cell viability with

increasing concentrations of all the leaf extracts of

Percentage inhibition (%)

Percentage inhibition of

ABTS radical cation (%)

100.0

90.0

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (Min)

Figure 1. Percentage inhibition of ABTS radical

cations in the presence of the extracts from the

leaves of Pereskia bleo (100 µg/mL of methanol,

ethyl acetate or aqueous extracts, 50 µg/mL of

t-butanol extract) over 12 minutes. The data are

expressed as the mean percentages inhibition of

ABTS radical cations ± standard deviation (S.D.).

denotes methanol extract at 100 µg/mL; ∆

denotes ethyl acetate extract at 100 µg/mL; ×

denotes t-butanol extract at 50 µg/mL; ◊ denotes

aqueous extract at 100 µg/mL.

100

90

80

70

60

50

40

30

20

10

0

0 20 40 60 80 100 120

Concentration (ug/mL)

Figure 2. Percentage inhibition of ABTS radical

cations by various concentrations of the extracts

from the leaves of Pereskia bleo at the sixth minute.

denotes methanol extract; ∆ denotes ethyl acetate

extract; × denotes t-butanol extract; ◊ denotes aqueous

extract. The linear regression equations for methanol

extract: y = 0.6668x + 8.9156; ethyl acetate extract: y

= 0.7867x + 0.9745; t-butanol extract: y = 1.5734x +

0.9745; aqueous extract: y = 0.683x + 14.789.

Jostt vol 6.indd 34 7/22/10 10:09:11 PM


P. bleo against both the 4T1 and 3T3 cells (Fig. 3).

However, the IC 50 values for all the extracts were

greater than the highest tested concentration (400

µg/mL). We could not use concentrations higher

than 400 µg/mL due to insolubility of these extracts

at high concentrations. The percentage cell viability

values of the 4T1 and 3T3 cells treated with 400

µg/mL of the various leaf extracts for 72 hours are

presented in Table 2. All the extracts resulted in a

greater reduction of the percentage viability of the

4T1 cells compared to the 3T3 cells.

Table 2. The percentage of viable 4T1 and 3T3 cultured

cells treated with 400 µg/mL of extract from the leaves of

Pereskia bleo for 72 hours.

Extract

% ± S.D. of viable cells

4T1 cells 3T3 cells

Methanol leaf extract 80.81 ± 0.97 107.55 ± 2.00

Ethyl acetate stem extract 72.63 ± 1.65 73.36 ± 7.76

t-Butanol leaf extract 78.55 ± 4.92 100.37 ± 3.57

Aqueous leaf extract 77.44 ± 2.10 105.89 ± 5.41

Figure 3. Percentage of viable (a) 4T1 cells and (b) 3T3

cells cultured in the presence of the methanol, ethyl

acetate, t-butanol and aqueous extracts of the leaves of

Pereskia bleo for 72 hours.The data are expressed as the

mean percentages of cell viability ± standard deviation

(S.D.). denotes methanol extract; ∆ denotes ethyl

acetate extract; × denotes t-butanol extract; ◊ denotes

aqueous extract; + denotes cisplatin.

Ability of leaf extracts of P. bleo to limit natural

apoptotic cell death in 3T3 cells

Figure 4 shows the enrichment factors calculated

when 3T3 cells were cultured in the presence and

absence of 50 µg/mL or 100 µg/mL extracts from the

leaves of P. bleo. The enrichment factor for untreated

cells (i.e. negative control) was 1.0. Enrichment

factor value less than 1.0 indicated a reduction in cell

death by apoptosis. All the leaf extracts were able to

reduce natural apoptotic cell death in 3T3 cells. The

ability to limit natural apoptotic cell death in 3T3

cells was greater at 50 µg/mL than at 100 µg/mL for

all extracts.

Ability of leaf extracts of P. bleo to limit oxidantinduced

apoptotic cell death in 3T3 cells

An increase in apoptotic cell death was observed in

the 3T3 cells cultured for 24 hours in the presence

of 10 µM DPPH (Fig. 5). However, the extent of

the DPPH-induced apoptotic cell death was reduced

when the cells were pre-incubated with extracts from

the leaves for one hour before DPPH were added to

the culture wells. There was no significant difference

in the level of oxidant-induced apoptotic cell death

when the 3T3 cells were cultured in the presence of

50 µg/mL of the leaf extract, compared to the higher

concentration used i.e.100 µg/mL. The protective

effects of all the extracts against oxidant-induced

cell death were greater than Trolox or vitamin C.

Figure 4. The ability of extracts from the leaves of

Pereskia bleo to limit natural apoptotic death in 3T3 cells.

The data are expressed as the mean enrichment factors

± standard deviation (S.D.). denotes cells in media

only; * denotes cells in media containing 0.4% DMSO;

◊ denotes aqueous extract; denotes methanol extract;

× denotes t-butanol extract; ∆ denotes ethyl acetate

extract; + denotes vitamin C; - denotes trolox.

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Journal of Science and Technology in the Tropics (2010) 6: 31-38

Among the extracts, the ethyl acetate extract showed

the highest protective effect against oxidant-induced

apoptotic cell death.

DISCUSSION

The sequential fractionation of the crude methanol

extracts of the leaves of P. bleo used in this study

showed that the bioactive compounds with antioxidant

activity were concentrated in the t-butanol extract

(416 µmole Trolox Equivalent per gram dry weight),

followed by the ethyl acetate extract. The methanol

and aqueous extracts had similar antioxidant

properties. These antioxidant properties are moderate

compared to those of commercial black teas and

green teas which have antioxidant capacity ranging

between 235 µmole Trolox Equivalent per gram dry

weight and 1526 µmole Trolox Equivalent per gram

dry weight [9]. The antioxidant effects of the extracts

are also much less than that of Vitamin C, a known

antioxidant compound, which has an antioxidant

capacity of 5158 Trolox Equivalent per gram dry

weight [9]. As such, the leaf of P. bleo cannot be

considered to be a rich source of antioxidants.

The IC 50 values for all the crude and fractionated

extracts of the leaves of P. bleo were greater than 400

µg/mL. The extracts could not be tested at higher

concentrations due to the lack of solubility. According

to the National Cancer Institute guideline, the crude

Figure 5. The ability of extracts from the leaves of

Pereskia bleo to limit DPPH-induced apoptotic death in

3T3 cells. The data are expressed as the mean enrichment

factors ± standard deviation (S.D.). o denotes cells in

media only; * denotes cells treated with DPPH; ◊ denotes

aqueous extract; denotes methanol extract; ×

denotes t-butanol extract; Δ denotes ethyl acetate

extract; + denotes vitamin C; - denotes trolox.

extract of a plant should have an IC 50 value of less

than 20 µg/mL in order to be considered as cytotoxic

against the treated cells [11]. The results suggested

that these extracts did not contain significant amount

of compounds with anti-proliferative properties.

Since the anti-proliferative activity of the fractionated

extracts (ethyl acetate, t-butanol and aqueous extracts)

are not greater than that of the crude methanol extract,

the methanol extract is unlikely to contain any

significant amount of anti-proliferative compounds.

A comparison with the reported anti-proliferative

activity of the stem extracts of P. bleo [6] shows

that the leaf extracts have higher anti-proliferative

activity. Moreover, the leaf extracts have a higher

anti-proliferative effect on the 4T1 mouse mammary

cancer cells compared to the normal 3T3 mouse

fibroblast cells. However, the reverse trend has been

reported for the stem extracts [6]. This indicates that

the leaf extracts of P. bleo might be a better target to

be developed as anti-cancer agent compared to the

stem extracts.

Treatment of the normal mouse fibroblast cells

(3T3) with the leaf extracts of P. bleo for 24 hours

resulted in a lower level of apoptosis in the cells

compared to that occurring in the non extracttreated

cells grown under the same conditions. The

results indicated that all the methanol, ethyl acetate,

t-butanol and aqueous extracts had the ability to

protect the 3T3 cells against natural programmed

cell death by apoptosis. The protective effects were

more obvious at 50 µg/mL compared to 100 µg/mL

of the extracts. The higher level of apoptosis at the

higher extract concentration was consistent with the

results of the anti-proliferation assay which showed

a gradual decrease in cell viability with increasing

extract concentration. All the fractionated extracts of

the leaves of P. bleo showed higher apoptosis-limiting

activities than the crude methanol extract, except

the aqueous extract. This suggested that the crude

methanol extracts contained bioactive component(s)

that could protect the normal mouse fibroblast

cells against natural apoptotic cell death, and these

components could be concentrated by fractionating

the crude methanol extracts.

The protective effects of the leaf extracts of P. bleo

against oxidant-induced cell death were also evaluated

by pre-treating the 3T3 cells with the extracts before

the oxidant, 2,2-Diphenyl-1-Picrylhydrazyl (DPPH),

was added. All the extracts were able to limit oxidantinduced

cell death, with the ethyl acetate extract being

Jostt vol 6.indd 36 7/22/10 10:09:17 PM


the most effective. The ability to limit oxidant-induced

cell death was not significantly different between 50

µg/mL and 100 µg/mL of the extracts, indicating that

the maximum effective concentration was reached.

The findings on the protective effects of the

extracts against oxidant-induced cell death were

consistent with the moderate antioxidant activities of

the extracts determined by the ABTS radical cation

scavenging assay. Nevertheless, the t-butanol extract

possessed the highest in vitro antioxidant activity,

whereas the ethyl acetate extract was more effective

in limiting oxidant-induced cell death. The ability of

the extracts to scavenge ABTS radical cation implied

that these extracts could act as reactive oxygen

species scavenger in diseases caused by oxidantinduced

cell death. An example of plants which

possess such property is Pogostemon cablin, a wellknown

Korean traditional medicine that has been

proven to be beneficial for patients with cerebral

stroke [12]. The water extract of P. cablin has been

shown to be able to protect the human neuroglioma

cells against necrotic and apoptotic cell death induced

by hydrogen peroxide. The protective effects of the

P. bleo extracts against oxidant-induced cell death

should be investigated further for their potential to

be developed for treatment of neurodegenerative

diseases which can be caused by reactive oxygen

species.

From this study, we can conclude that all of

the crude methanol and fractionated ethyl acetate,

1. Goh K.L. (2000) Malaysian Herbaceous Plants.

Millennium Edition. Advanco Press, Malaysia.

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3.

4.

5.

Tan M.L., Sulaiman S.F., Najimuddin N., Samian

M.R. and Tengku Muhammad T.S. (2005) Methanolic

extract of Pereskia bleo (Kunth) DC. (Cactaceae)

induces apoptosis in breast carcinoma, T47-D cell

line. Journal of Ethnopharmacology 96: 287-294.

Er H.M., Cheng E. and Radhakrishnan A.K.

(2007) Anti-proliferative and mutagenic activities

of aqueous and methanol extracts of leaves from

Pereskia bleo (Kunth) DC (Cactaceae). Journal of

Ethnopharmacology 113: 448-456.

Abd Malek S.N., Abdul Wahab N., Yaacob H., Sim

K.S., Hong S.L., Lee G.S. and Rahman S.N.S.A.

(2008) Cytotoxic activity of Pereskia bleo (Cactaceae)

against selected human cell lines. International

Journal of Cancer Research 4: 20-27.

Wahab S.I.A., Abdul A.B., Mohan S.M., Al-Zubairi

A.S., Elhassan M.M. and Ibrahim M.Y. (2009)

REFERENCES

t-butanol and aqueous extracts of the leaves of P.

bleo do not have significant anti-proliferative effect

on both the mouse mammary cancer cells (4T1) and

the normal mouse fibroblast cells (3T3). They are not

cytotoxic, based on the criterion that a crude extract

should have an IC 50 of less than 20 µg/mL for it to

be considered cytotoxic against the treated cells [11].

Nevertheless, the leaf extracts exhibited selectivity in

inhibiting the proliferation of the mouse mammary

cancer cells as opposed to the normal mouse fibroblast

cells. One possible mechanism for the selective killing

of the cancer cells may be related to the ability of the

extracts to activate the p53 function in tumour cells,

causing their growth arrest or apoptosis, as reported

in Ashwagandha (Withania somnifera), a herb

commonly used in Ayurvedic medicine [13]. Among

the extracts tested, the t-butanol extract of the leaves

of P. bleo possesses the highest antioxidant property.

Yet, the antioxidant capacity is only moderate when

compared to other known antioxidant substances

such as green tea leaves and vitamin C. The ability of

the extracts of the leaves of P. bleo in limiting natural

and oxidant-induced cell death in normal mouse

fibroblast cells suggests that the plant may be useful

as a remedy for diseases related to oxidative stress.

Acknowledgement – We would like to thank Taman

Pertanian Malaysia, Universiti Putra Malaysia, for kind

donation of the Pereskia bleo plant. This work was supported

by a grant from the International Medical University.

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Journal of Science and Technology in the Tropics (2010) 6: 39-42

Abdominal colour polymorphism in female Asian Golden Web Spider

Nephila antipodiana (Araneae: Nephilidae)

Yong Hoi Sen1 , Rosli Hashim1 , Daicus Belabut1 and Lim Phaik Eem1,2 1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

2Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

(E-mail: yong@um.edu.my)

Abstract Nephila antipodiana (Batik Golden Web Spider) occurred in large numbers in several localities in

Kelantan and Terengganu, east coast of Peninsular Malaysia. Three abdominal (opisthosomal) colour morphs

– yellow, greenish-yellow and reddish-brown – were present in adult female spiders. Only two colour morphs

appear to be present in a particular locality. The yellow morph occurred in all the localities investigated. Of

the two non-yellow morphs, the reddish-brown morph was found only in one locality while the greenish-yellow

morph was more widespread. The present study does not show unequivocally the association of colouration

with habitat usage. Whether yellow colour in N. antipodiana confers a selective advantage remains to be

verified. In addition to opisthosomal colour polymorphism, the colour and number of spots (or sigillae) on the

dorsal surface of the abdomen of female N. antipodiana are also variable. The juvenile spiders have different

colour pattern from the adults.

Keywords polymorphism – golden web spider – Arachnida – Malaysia – opisthosoma colour

INTRODUCTION

The Batik (or Asian) Golden Web Spider Nephila

antipodiana (Walckenaer 1842) was first reported

in Peninsular Malaysia (and Malaysia) in 2009 [1].

It was originally described as Epeira antipodiana

Walckenaer 1842. It is distributed in China, Philippines

to New Guinea, Solomon Is., and Queensland [2]. In

Southeast Asia it has been recorded in Singapore,

Indonesia, Thailand and the Philippines [3, 4]. It has

also been found in Borneo (Joseph K.H. Koh, pers.

comm.).

It is evident from the literature that N. antipodiana

is a variable species [1, 3-5]. In the Philippines the

dorsal colour of the abdomen (opisthosoma) in the

female is yellow, with six pairs of subovate yellow

spots dorsally arranged longitudinally in rows, each

spot with a thin black margin [4]. The abdomen of

the female spider in Australasia is pale yellow, with

darker markings on anterior and posterior margins,

and without large yellow spots [5]. In Singapore the

dorsal colour of the abdomen may be yellowish green

[3] or reddish brown [1] with yellow spots.

Although abdominal (opisthosomal) colour

variation occurs in the species, as evidenced in

the different colour morphs, there is no report on

polymorphism. We report here the occurrence of

polymorphism in the dorsal abdominal colour of

adult female N. antipodiana in Peninsular Malaysia.

MATERIALS AND METHODS

A field survey of N. antipodiana was carried out

from 12-15 February 2010 in Kelantan and

Terengganu, Peninsular Malaysia. The dorsal colour

(yellow, greenish-yellow, and reddish-brown) of the

abdomen of every adult female spider in a chosen

locality was recorded. The number of juvenile and

associated male individuals was also recorded.

Five localities were studied – four in Kelantan

(Pantai Melawi and Sungai Dua in Bachok, Tok

Bok in Machang, and Gua Musang highway) and

one in Terengganu (Lata Belatan). Pantai Melawi

consisted of eight patches which were separated

although not far from each other.

Bachok is situated near the coast, with typical

Malay village setting. Both Machang and Gua

Musang are in the interior. Lata Belatan is a forest

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Journal of Science and Technology in the Tropics (2009) 5: 125-131


40

Journal of Science and Technology in the Tropics (2010) 6: 39-42

recreation park – the study was carried out along the

road bordered by plantation.

Representative specimens were collected and

preserved in ethanol. Photographs were also taken of

the various colour morphs and life stages.

RESULTS

Three principal abdominal colour morphs – yellow

(Figs. 1, 2), greenish yellow (Fig. 3) and reddish

brown (Fig. 4) – were observed in the adult female

N. antipodiana. Their occurrence and abundance in

four localities in Kelantan and one in Terengganu are

summarized in Table 1. The juveniles (Fig. 5) and

males were not included as the former had different

colour pattern and the male did not show colour

variation.

The yellow morph was present in all the five

localities. Of the other two morphs, the greenishyellow

morph was found only in Kelantan while the

reddish-brown morph was observed in Lata Belatan,

Terengganu. The frequency of the yellow morph

ranged from 7.69% in Sungai Dua, Bachok to 25.71%

in Lata Belatan, Terengganu (Table 1).

DISCUSSION

Colour and pattern variation in spiders has been

widely investigated [6]. To-date three major classes

of pigments – ommochromes (yellow, red, brown),

bilins (blue, green) and guanine (white, silver) – have

been identified. The earlier work before 1998 on the

evolution and ecology of spider colouration has been

well reviewed [6]. Although polymorphisms have

been reported for a number of species, the genetic

bases have generally not been elucidated. In general,

sex linkage appears to be absent but sex limitation

is quite common. It is acknowledged that the

manifestation of spider colouration is very complex,

involving a host of possible factors. Various functions

have been suggested – cryptic/disruptic, mimetic and

aposematic as well as thermoregulatory.

The Hawaiian happy-face spider, Theridion

grallator Simon (Therididae) provided the most

spectacular example of colour polymorphism in

spiders. In this spider, the opisthosomal colour may

be controlled by simple Mendelian alleles at a single

autosomal locus or multiple closely linked loci.

The plain yellow morph is recessive to the other

colour morphs [7, 8]. More recently, another species

Theridion californicum Banks has been found to

exhibit an equally extraordinary visible colour

and pattern polymorphism [9]. The polymorphism

comprises one common morph (Yellow) and at least

ten relatively rare patterned morphs, with Yellow

probably recessive to all other morphs.

In the present study, the yellow morph of female N.

antipodiana occurred in all the localities investigated

(Table 1). Lata Belatan had the highest percentage

(25.71%). This locality had denser vegetation, being

in a plantation area. In Gua Musang highway, an

open area, the percentage was 16.98%. Although it

has been suggested that yellow colour is most cryptic

in the below-leaf environment and hence selected for

Table 1. Abundance of abdominal colour morphs in adult female Nephila antipodiana from

four localities in Kelantan and Terengganu, east coast of Peninsular Malaysia, recorded in

February 2010.

Locality Greenish Yellow Yellow Reddish Brown % Yellow

Bachok: Pantai Melawi

1 outside chalet

2 coconut palms

3 house compound

4 waste land

5 coconut palms

6 Hibiscus tiliaceus

7 coconut palms

8 open space

Total (1 – 8)

2

28

39

18

26

33

8

7

161

2

3

1

5

6

2

19

50.00

9.68

0

0

3.70

13.16

42.86

22.22

10.56

Bachok: Sungai Dua 24 2 7.69

Machang: Tok Bok 82 10 10.87

Gua Musang: highway 44 9 16.98

Terengganu: Lata Belatan 27 78 25.71

Jostt vol 6.indd 40 7/22/10 10:09:19 PM


Figures 1-4. Abdominal colour morphs of adult female Nephila antipodiana (photos: H.S. Yong).

1. Yellow morph; 2. Venter of a Yellow morph; 3. Greenish-yellow morph; 4. Reddish-brown morph.

[9], the present study does not show unequivocally

the association of colouration with habitat usage.

Whether yellow colour in N. antipodiana confers

a selective advantage remains to be verified. In

Pantai Melawi with a mixture of micro-habitats, the

percentage of yellow morph ranged from 9.68% to

42.86% in patches with coconut palms while in an

open space it was 22.22% in contrat to 13.16% in an

area with Hibiscus tiliaceus (Table 1).

It is noteworthy that in the present study only two

colour morphs appear to be present in a particular

locality (Table 1). Of the two non-yellow morphs, the

reddish-brown morph was found only in one locality

(Lata Belatan) whereas the greenish-yellow morph

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41

Journal of Science and Technology in the Tropics (2010) 6: 39-42


42

Journal of Science and Technology in the Tropics (2010) 6: 39-42

was more widespread. The reddish-brown morph

was found in Teluk Chempedak, Pahang where N.

antipodiana was first reported for Peninsular Malaysia

[1]. In this locality, a yellow individual had been

observed in the hill forest (H. S. Yong, unpublished

information). At the University of Malaya, a single

individual of the greenish morph had been found

among ornamental palms (H. S. Yong, personal

observation). The significance of the presence of only

a single non-yellow morph in a particular locality

remains to be elucidated.

In addition to opisthosomal colour polymorphism,

the colour and number of spots (or sigillae) on the

dorsal surface of the abdomen of N. antipodiana are

also variable. The dorsum may be marked with spots

or sigillae. In the Philippines, the yellow morph has

1. Yong H.S. (2009) Nephila antipodiana (Araneae:

Nephilidae) from Pahang: a new record for

Peninsular Malaysia. Journal of Science and

Technology in the Tropics 5: 19-21.

2. Platnick N.I. (2010) The world spider catalog, version

10.5. American Museum of Natural History, online

at http://research.amnh.org/entomology/spiders

/catalog/index.html.

3. Koh J.K.H. (1989) A guide to common Singapore

spiders. Singapore Science Centre, Singapore.

4. Barrion A.T. and Litsinger J.A. (1995) Riceland spiders

5.

6.


 


Figure 5. Juvenile female Nephila antipodiana with

different colour pattern from adult female spider. (photo:

H.S. Yong)

of South and Southeast Asia. CAB International,

Wallingford, UK.

Harvey M.S., Austin A.D. and Adams M. (2007) The

systematic and biology of the spider genus Nephila

(Araneae: Nephilidae) in the Australasian region.

Invertebrate Systematics 21: 407-451.

Oxford G.S. and Gillespie R.G. (1998) Evolution

REFERENCES

six pairs of subovate yellow spots, each with a thin

black margin [4]. In Australasia, the dorsal surface

has four pairs of sigillae [5]. The yellow morph in the

present study (Peninsular Malaysia) is characterized

by five pairs of yellow spots/sigillae, each well

defined by black margin. There may also be a sigilla/

spot situated at the middle of the dorsal surface (Fig.

1).

The yellow morph of N. antipodiana in general

appears to possess fewer spots than the non-yellow

morphs. In the present study, the yellow morph had

five pairs of spots/sigillae. On the other hand, the

greenish-yellow and reddish-brown morphs had nine

pairs of spots which were variable in size. Another

difference is the spots in the yellow morph tended

to assume the appearance of sigillae – depression at

least at the posterior part of the spot. The intensity

and dimension of the black outline are also variable.

Spots in the greenish-yellow and reddish-brown

morphs are not marked by black outline.

In sum, N. antipodiana is highly variable

in colouration and pattern/marking. Cursory

observations indicate the presence of other colour

morphs. Detailed studies are needed to elucidate the

ontogeny of colour and pattern development, and

the function and mechanism of the variation and

polymorphism. N. antipodiana could be an excellent

model for evolutionary and ecological studies.

Acknowledgements – We thank the University of

Malaya for financial and other supports to carry out this

study.

7.

8.

9.

and ecology of spider coloration. Annual Review of

Entomology 43: 619-643.

Gillespie R.G. and Tabashnik B. (1989) What makes

a happy face? Determinants of colour pattern in the

spider Theridion grallator (Araneae, Theridiidae).

Heredity 62: 335-363.

Oxford G.S. and Gillespie R.G. (1996) Genetics of a

colour polymorphism in Theridion grallator (Araneae:

Theridiidae), the Hawaiian happy-face spider, from

Greater Maui. Heredity 76: 238-248.

Oxford G.S. (2009) An exuberant, undescribed

colour polymorphism in Theridion californicum

(Araneae, Theridiidae): implications for a theridiid

pattern ground plan and the convergent evolution of

visible morphs. Biological Journal of the Linnean

Society 96: 23-34. With 2 figures.

Jostt vol 6.indd 42 7/22/10 10:09:22 PM


Journal of Science and Technology in the Tropics (2010) 6: 43-47

Scaled down operation of the United Nations University/International Centre

for Theoretical Physics Plasma Focus Facility (UNU/ICTP PFF) as an

extreme ultraviolet source

Rattachat Mongkolnavin11* 1 2

, Prajya Tangitsomboon and Chiow San Wong

1Department of Physics, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

2Plasma Research Laboratory, Physics Department, Faculty of Science, University of Malaya,

50603 Kuala Lumpur, Malaysia

(*E-mail: rattachat.m@chula.ac.th)

Abstract The possibility of operating the 3.3 kJ United Nations University/International Centre for Theoretical

Physics Plasma Focus Facility (UNU/ICTP PFF) with a scaled down electron temperature so as to tune the

emission spectrum to the EUV region is being considered. In particular, we investigated the scaled down

operation of this plasma focus device from its original 3.3 kJ electrical input energy to as low as 960 J.

Experiments show that for discharges performed at 8 kV and with a shortened electrode length of 9 cm from

the original 16 cm, the emission was predominantly in the wavelength range of 12 to 18 nm.

Keywords plasma focus – EUV source

INTRODUCTION

The development of Extreme Ultraviolet (EUV)

radiation sources is recently gaining much interest

in semiconductor manufacturing industry due to the

expectation that the Next Generation Lithography

(NGL) will be using the wavelength of 13.5 nm [1].

Many types of EUV radiation sources, including the

laser produced plasma and pulsed discharge sources

such as the capillary discharge [2, 3], vacuum spark

[4, 5] and plasma focus [6, 7] are being considered

by researchers worldwide. These radiation sources,

especially the pulsed discharge sources are favourable

as EUV radiation source because of their lower cost

and simplicity in operation when compared to other

radiation sources.

The United Nations University/International

Centre for Theoretical Physics Plasma Focus Facility

(UNU/ICTP PFF) is a 3.3 kJ Mather type plasma

focus [8] which is optimized to produce fusion

neutrons with deuterium as the operating gas [9].

With argon or xenon as the working gas, it is known

to be capable of producing intense radiation in the

X-ray region [10-12]. The typical plasma produced

by the optimized UNU/ICTP PFF has an electron

temperature of several keV and an expected electron

density of greater than 10 19 cm -3 [8].

In this work, we consider the possibility of scaling

down the temperature of the UNU/ICTP PFF argon

plasma focus so as to operate it as an EUV radiation

source. The scaling can be done in two approaches.

The first approach is by reducing the electrical input

energy or by increasing the operating pressure while

the original geometry of the UNU/ICTP PFF is

maintained. In this case, the dynamics of the current

sheet is slowed down so that the focusing occurs

at a time after the peak of the discharge current for

electrodes with original length of 16 cm. This will

effectively make the focus less efficient although

the required electron temperature of around 110 eV

can be obtained. In the second approach, the reduced

efficiency caused by the mismatch of the dynamic

characteristic time and the electrical characteristic

time can be compensated by reducing the length of

the electrodes. A discharge voltage of as low as 6.5

kV corresponding to an electrical input energy of 634

J is shown to be sufficient.

According to the Coronal Equilibrium Model

(CEM), there are two possible ranges of temperature

at which argon plasma is expected to consist of

Jostt vol 6.indd 43 7/22/10 10:09:23 PM

43


44

Journal of Science and Technology in the Tropics (2010) 6: 43-47

ionic species that can be considered as emitters of

line radiations at wavelength around 13.5 nm. At a

temperature of around 10 to 20 eV, the Ar 6+ and Ar 7+

are prominent, whereas at a temperature of around

100 to 130 eV, the argon plasma is expected to consist

of predominantly the Ar 10+ and Ar 11+ ionic species.

The population distribution of these two groups of

ionic species as predicted by the CEM is illustrated

in Figure 1. These ionic species are known to be able

to emit intense line radiations at or near 13.5 nm as

listed in Table 1 [13].

In order to test the possibility of tuning the UNU/

ICTP PFF to produce condition which is sufficient

to produce intense EUV radiation but not X-ray, we

scaled down the operating parameters, particularly

the input electrical energy. In order to match the

slower dynamics of the current sheet due to the

lowering of the input energy, the electrode lengths of

the device were reduced to 9 cm and 7.5 cm and the

discharge voltage was reduced to 8 kV and 6.5 kV

respectively, corresponding to input energy of 960 J

and 634 J respectively. The operating pressure was

Table 1. Expected lines near 13.5 nm from argon ions Ar 6+ ,

Ar 7+ , Ar 10+ and Ar 11+ .

Ion Wavelength

(nm)

Transition Upper

level

Lower

level

Ar 6+ 13.4797 5p-3s (3s5p) 1 P 1 (3s 2 ) 1 S 0

Ar 7+ 13.5591 9f-4d (9f) 2 F 7/2 (4d) 2 D 5/2

Ar 10+ 13.57 2p-2s (2s2p 5 ) 1 P 1 (2p 4 ) 3 P 1

Ar 10+ 13.563 2p-2s (2s2p 5 ) 1 P 1 (2p 4 ) 3 P 1

Ar 11+ 13.6 2p-2s (1s 2 2p 5 ) 2 P 3/2 (2s2p 4 ) 4 P 3/2

Population

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Ar 6+

Ar 7+

Ar 10+

Ar 11+

50 100 150 200

Temperature (eV)

Figure 1. The temperature range at which argon

ions Ar 6+ , Ar 7+ , Ar 10+ and Ar 11+ are prominent

predicted by Coronal Equivalent Model.

adjusted within the range of 0.5 to 2.5 mbar for fine

tuning. Focusing action was obtained over this range

of pressure and the radiation emission in the EUV

region was observed consistently.

EXPERIMENTAL SETUP

The experimental setup is shown schematically in

Figure 2. The electrode system consisted of a central

hollow copper anode of 1.9 cm diameter, and a

cathode of six copper rods arranged in a circle of 6.4

cm diameter concentric with the anode. The anode

was insulated from the cathode at the back-wall by

a Pyrex glass tube. Three anode lengths were used,

including the original length of 16 cm, and two other

shorter lengths of 9 cm and 7.5 cm. The plasma focus

was operated with argon at pressures in the range of

0.5 to 2.5 mbar. The discharge voltage was varied

from 6 kV to 12.5 kV. The diagnostics used included

the Rogowski coil for discharge current measurement,

resistive voltage divider for transient voltage

measurement, PIN Si-diodes for X-ray measurement,

and silicon p-n junction photodiode with integrated

multilayer thin film filter for EUV measurement.

The BPX65 PIN Si-diode operated with its glass

window removed is now commonly used for the

measurement of soft X-ray from pulsed plasma due

to its fast rise-time of 2 ns [10, 12]. BPX65 diode

purchased off the shelf has a typical spectral response

of above 0.1 A/W in the wavelength range of 450 nm

to 1050 nm, with a peak spectral response of 0.55 A/W

at 900 nm [11]. However, with the front glass window

of its TO-18 casing removed, its spectral response can

be extended to the X-ray region. In order to exclude

photons in the visible region, the diode was covered

by 24 µm aluminized Mylar.

+ HV

BPX 65

SXUV

Anode

Insulator

Cathode

Figure 2. Schematics showing the experimental setup.

Jostt vol 6.indd 44 7/22/10 10:09:24 PM


A silicon p-n junction photodiode with 100 nm

silicon and 200 nm zirconium directly deposited

filter [15] was used to measure EUV radiation

in the wavelength range of 12 nm to 18 nm. The

responsivity curves of the modified BPX65 covered

with 24 µm aluminized Mylar and SXUV diode with

100 nm Si and 200 nm Zr multilayer filter, plotted on

the same graph, are shown in Figure 3. It can be seen

that the filtered BPX65 was responsive to photons

with wavelength below 1 nm, with peak response of

0.147 A/W at wavelength of 0.35 nm. On the other

hand, the silicon p-n junction diode had a clear pass

band in the range of 12 to 18 nm, with peak response

of 0.095 A/W at wavelength of 12.8 nm. While the

visible range was clearly rejected by the filter, the

response of this diode to photons in X-ray region

was not certain. In the experiments described here,

the filtered BPX65 and the filtered SXUV were used

to measure the radiation emitted from the plasma

simultaneously. They were mounted to view the

Responsivity (A/W)

Voltage (kV)

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

BPX65 with 24 µm Aluminized Mylar

SXUV filtered by 100 nm Si & 200 nm Zr

2 4 6 8 10 12 14 16 18 20

Wavelength (nm)

Figure 3. The responsivity curves of BPX65

PIN diode (with 24 µm aluminized Mylar)

plotted together with that of SXUV p-n junction

diode with 100 nm Si and 200 nm Zr.

4

2

0

Current

Voltage

-100

0.0 1.0x10 -6 2.0x10 -6 3.0x10 -6 4.0x10 -6 5.0x10 -6 6.0x10 -6 7.0x10 -6

-2

Time (s)

Figure 4. The typical current and voltage signals

of a plasma focus discharge.

200

150

100

50

0

-50

Current (kA)

plasma in the side-on location at a distance of 30 cm

from the axis of the electrode system. This allowed

us to identify without ambiguity the spectral range of

the radiation detected.

RESULTS AND DISCUSSION

In its original design, the UNU/ICTP PFF had an

electrode length L = 16 cm. For a typical focusing

discharge at discharge voltage V = 12.5 kV and at an

operating pressure P = 0.5 mbar argon, the signature

voltage spike and current dip (Fig. 4) are consistently

obtained. Under this operating condition, the current

sheet in the axial acceleration phase of the plasma

focus dynamics achieves an average speed of about

4 cm µs -1 . The X-ray emission characteristics of the

plasma produced under similar condition had been

studied and reported before [7, 9]. In this study, the

measurement of the emission was extended to the

EUV region by using a SXUV silicon p-n junction

photodiode together with a BPX65 PIN diode. The

signals obtained, together with the voltage spike of a

typical 12.5 kV discharge, operated at 0.5 mbar argon

of the UNU/ICTP PFF with an electrode length of 16

cm are shown in Figure 5

The X-ray emission for a typical focusing

discharge of the UNU/ICTP PFF at V = 12.5 kV, P

= 1.5 mbar argon and with L = 16 cm is as shown

in Figure 5. This X-ray pulse is similar to those

observed in many plasma focus devices as reported

in the literature [10-12]. It indicates the hottest phase

of the plasma produced. The occurrence of the hottest

phase of the plasma coincided with the peak of the

voltage spike. Correspondingly, the SXUV diode

registered a large EUV pulse. From the area under

SXUV (V) Voltage (kV)

30

25

20

15

10

5

0

3.0x10 -6

-5

40

30

20

10

0

-10

3.0x10 -6

-20

4.0x10 -6

4.0x10 -6

SXUV

BPX65

5.0x10 -6

5.0x10 -6

Time(s)

6.0x10 -6

6.0x10 -6

7.0x10 -6

20

10

0

7.0x10 -6

Figure 5. The voltage, EUV and X-ray signals obtained

simultaneously for a 12.5 kV discharge of a plasma focus

with electrode length L = 16 cm and operated with 1.5

mbar argon.

Jostt vol 6.indd 45 7/22/10 10:09:25 PM

BPX65 (V)

45

Journal of Science and Technology in the Tropics (2010) 6: 43-47


46

Journal of Science and Technology in the Tropics (2010) 6: 43-47

this EUV pulse, the total photon energy emitted from

the plasma (assuming isotropic source) in the range

of 12 to 18 nm could be estimated to be about 50 mJ.

While the detection of the X-ray pulse for the 12.5

kV discharge is known to indicate the formation of a

focused plasma with temperature of probably greater

than 1 keV, the absence of X-ray radiation in a 8 kV

discharge is believed to indicate a lower temperature

plasma. The set of similar signals (Fig. 5) obtained

for an 8 kV discharge at 0.5 mbar argon is shown in

Figure 6. In this discharge, the length of the electrodes

of the UNU/ICTP PFF was shortened to 9 cm. This

discharge displays several features of its radiation

emission which are distinctly different from that of

the 12.5 kV discharge. Firstly, no X-ray was detected

for this discharge indicating lower temperature

plasma. The voltage signal showed multiple spikes

with low amplitudes. The corresponding EUV signal

also consisted of multiple pulses which occurred over

duration of more than 2 µs. The peak amplitude of the

pulse, however, was much lower than those observed

for discharges at 12.5 kV. However, due to long pulse

width, the area under this pulse corresponded to total

photon energy of about 100 mJ. The input energy of

the UNU/ICTP PFF was further tuned down to 540

J by operating at condition of V = 6.5 kV and L =

7.5 cm. The output EUV pulse was insignificantly

low as compared to those operated at 8 kV discharge

voltage.

In order to fine tune the EUV output from the

plasma focus at the three discharge voltages tested,

namely V = 12.5 kV, 8 kV and 6 kV, the operating

SXUV (V) Voltage (kV)

5

4

3

2

1

0

-1

3.0x10 -6

5

4

3

2

1

0

-1

3.0x10 -6

4.0x10 -6

4.0x10 -6

SXUV

BPX65

5.0x10 -6

5.0x10 -6

Time(s)

6.0x10 -6

6.0x10 -6

7.0x10 -6

5

4

3

2

1

0

-1

7.0x10 -6

Figure 6. The voltage, EUV and X-ray signals

obtained simultaneously for a 8 kV discharge of a

plasma focus with electrode length L = 9 cm and

operated with 0.5 mbar argon.

BPX65 (V)

pressure was varied from 0.5 mbar to 2.5 mbar for

a series of discharges. From the area under the EUV

pulse, the total photon energy emitted in the range of

12 to 18 nm was estimated and plotted (Fig. 7). It can

be seen that discharges with higher input energy of

2.3 kJ was able to produce EUV pulses with higher

amplitudes and shorter duration, but the total energy

corresponding to the photons emitted within the

spectral range of 12 to 18 nm was lower than those

emitted from discharges at 960 J input energy. EUV

energy of up to 100 mJ can be obtained from a 8 kV

discharge operated with 0.5 mbar argon.

CONCLUSION

By adjusting the operating parameters of the UNU/

ICTP PFF operated with argon from its originally

designed values of discharge voltage V = 15 kV,

electrode length L = 16 cm and P = 0.5 to 2.5 mbar

argon, we have obtained the possible experimental

conditions to operate it to produce plasmas with

conditions sufficient for emission up to the EUV

spectral range of 12 to 18 nm. The radiation energy

corresponding to this spectral range emitted from

a discharge with V = 12.5 kV, L = 16 cm and P =

0.5 to 2.5 mbar may be up to 95 mJ. This emission,

however, is accompanied by emission of X-ray

photons in the wavelength range of 0.3 to 3 nm. The

plasma produced under this condition is expected

to have achieved an electron temperature of up to

several keV. However, when the same plasma focus

device is operated at a scaled down condition of V

Figure 7. The variation of output EUV energy for

three sets of operation condition of discharge voltage

V and electrode length L at various pressure P. (i) V

= 12.5 kV, L = 16 cm; (ii) V = 8 kV, L = 9 cm; and (iii)

V = 6.5 kV, L = 7.5 cm.

Jostt vol 6.indd 46 7/22/10 10:09:27 PM


= 8 kV, L = 9 cm and with operating pressure in the

range of P =0.5 to 2.5 mbar argon, up to 102 mJ of

EUV photon energy can be produced and in this case,

no X-ray photon is produced. We expect the electron

temperature achieved in this case to be in the range of

several tens electron-volts. If one is currently having

the UNU/ICTP PFF and intend to use it as an EUV

source to test for the effect of EUV on a substract

which has good absorption in both the X-ray and

EUV regions, it may be necessary to eliminate the

X-ray component of the emission. In this case the

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Hoffman J.R. (2003) Proc. SPIE 5037: 807.

Rawat R.S., Zhang T., Phua C.B.L., Then J.X.Y.,

Chandra K.A., Lin X., Patran A. and Lee P. (2004)

Plasma Sources Sci. Technol. 13: 569.

REFERENCES

scaled down operation of the existing UNU/ICTP

PFF may be a possible solution. With this possibility

demonstrated, it is still necessary to point out that the

device can be further tuned to obtain the condition for

optimum EUV output.

Acknowledgements – The authors would like to extend

their great appreciation to Asian African Association for

Plasma Training (AAAPT) for its support to initiate plasma

focus research in this laboratory and to the Graduate

School, Chulalongkorn University for financial support.

8. Lee S., Tou T.Y., Moo S.P., Eissa M.A., Gholap A.V.,

Kwek K.H., Mulyodrono S., Smith A.J., Suryadi S.,

Usada W. and Zakaullah M. (1988) Am. J. Phys. 56:

62.

9. Yap S.L., Wong C.S., Choi P., Dumitrescu C. and Moo

S.P. (2005) Jpn. J. Appl. Phys. 44: 8125.

10. Favre M., Lee S., Moo S.P. and Wong C.S. (1992)

Plasma Sources Sci. Technol. 1: 122.

11.

Mohammadi M.A., Verma R., Sobhanian S., Wong

C.S., Lee S., Springham S.V., Tan T.L., Lee P. and

Rawat R.S. (2007) Plasma Sources Sci Technol. 16:

785.

12. Ng C.M., Moo S.P. and Wong C.S. (1998) IEEE Trans.

Plasma. Sci. 26: 1146.

13. http://Spectr-w3.snz.ru

14.

http://129.105.69.13/datasheets/Optoelectronics/

Photodiode_UDT_catalog.pdf

http://www.ird-inc.com

Jostt vol 6.indd 47 7/22/10 10:09:28 PM

15.

47

Journal of Science and Technology in the Tropics (2010) 6: 43-47


Journal of Science and Technology in the Tropics (2009) 5: 133-139

Jostt vol 6.indd 48 7/22/10 10:09:28 PM


Journal of Science and Technology in the Tropics (2010) 6: 49-52

An atmospheric pressure non-thermal plasma jet in nitrogen for

surface modification of polyethylene

D. P. Subedi1 , R. B. Tyata1 , A. Shrestha1 , D. Baral1 , D. K. Madhup1 and C. S. Wong2 1Department of natural Sciences, School of Science, Kathmandu University, Nepal

2 Plasma Research Laboratory, Department of Physics, University of Malaya, 50603 Kuala Lumpur,

Malaysia

Abstract In this paper, an atmospheric pressure plasma jet (APPJ) in nitrogen has been set up for possible

application in polymer surface modification. The effect on the hydrophilicity of a polyethylene (PE) surface

exposed to the plasma jet was investigated for different exposition times and distances from the nozzle. It has

been confirmed that the jet can modify polymer film with a work distance of more than 60 mm.

Keywords non-thermal plasma jet – surface modification – polyethylene – polymer film – hydrophilicity

INTRODUCTION

In the last one decade, research on non-thermal

plasma at atmospheric pressure has become a subject

of great interest because of its applications in various

fields. The characteristic of these types of plasma is

the existence of thermal non-equilibrium between the

electrons and the ions. The treatment of materials

by non-thermal atmospheric pressure plasma is a

promising technology that is simple to set-up, easy

and economical to operate and does not require

vacuum equipments.

Among the various applications of the nonthermal

atmospheric pressure cold plasmas are

sterilizations of living tissue without damage and

blood coagulation [1], modulation of cell attachment

[2], biological and chemical decontamination [3-5],

water decontamination and pollution control [6,7],

nanotechnology [8], surface cleaning, etching, thin

film deposition, surface modification and material

processing [9-13]. Atmospheric pressure plasma can

be generated by various methods: corona, glow, arc,

dielectric barrier discharge (DBD), RF discharge

and microwave discharges. The main problem with

these systems is that the working space is often

limited because of the electrode configuration. In

an atmospheric pressure plasma jet, the plasma

constituents are expelled through an orifice by a gas

flow which makes it possible for the treatment of large

objects. Forster et al. [14] reported an atmospheric

pressure plasma jet in a DBD configuration. This type

of plasma can be operated under high gas flow rate.

In 2003, Toshifuji et al. [15] reported a relatively cold

arc plasma jet produced under atmospheric pressure

having potential application for surface modification.

Quite recently, Takemura et al. [16] developed an

atmospheric pressure plasma jet with long flame which

can modify polymer film with a work distance of

over 200 mm. Recently, a double layered atmospheric

pressure plasma jet had also been reported [17].

In the present study, a non-thermal nitrogen

plasma jet was generated using a high voltage

power supply with output frequency of 10 to 30 kHz

under atmospheric pressure. The application of this

plasma jet to treat surface of polymer material is also

demonstrated.

EXPERIMENTAL SETUP

The schematic diagram of the plasma jet system

is depicted in Figure 1. A brass rod of diameter 3

mm was placed inside a glass tube of inner diameter

4 mm and outer diameter 6 mm. At one end of the

glass tube a steel cap electrode (9 mm diameter, 0.5

mm thickness and 20 mm long) with an orifice of

2 mm diameter, was mounted. The inner electrode

was connected to high voltage (0-20 kV) and high

frequency (10-30 kHz) power supply and the outer

electrode was grounded.

In this study, nitrogen is used as the working gas.

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Journal of Science and Technology in the Tropics (2009) 5: 133-139


50

Journal of Science and Technology in the Tropics (2010) 6: 49-52


Digital
Oscilloscope


N2


In order to evaluate the effectiveness of the plasma

jet in modifying the PE surface, hydrophilicity test

was performed. The contact angle of water droplets

at the surface of the PE film was measured using a

rame’-hart Contact Angle Goniometer. This unit

was equipped with standard software to analyze the

drop image for the calculation of surface energy.

The system offered a high level of computer aided

precision in measuring contact angle and therewith

facilitating the calculation of surface energy using

different model equation.

The water droplet contact angles on the surface

of PE films treated at different distances from the

nozzle of the jet and for different exposure times were

measured. Each contact angle presented in this paper

was an average of at least four measurements made

on different positions on the surface of PE.

RESULTS AND DISCUSSION

PC.
DAQ.


Voltage
Probe


Current
Probe


An example of the plasma jet obtained is shown in

Figure 2. A discharge between the centre electrode

and the steel cap containing the orifice is expected to

be produced and the jet is formed due to the flowing

nitrogen stream. The electron temperature of the

plasma near to the orifice is expected to be several

electron-volts. However, the temperature of the ions

and the neutral atoms/molecules is relatively cold and

was measured by using an IR thermometer to be in

the range of 28°C to 30°C. The length of the jet was

found to be dependent on the gas flow rate (Fig. 3).

The flow rate of nitrogen was varied from 1 to 5

litres per minute. To measure the length of the jet,

a scale was placed behind the jet while taking the

Arc
Plasma
Jet


Emission


Spectrometer


Figure 1. Experimental setup of the plasma jet.

Figure
1.
Experimental
setup
of
the
plasma
jet.


High
Frequency


Power
Supply


image of the jet by a digital camera. The lengths were

then determined from the photograph. It is clear that

the length of the jet increases proportionally with the

gas flow rate at the beginning and then levels off after

4 litres per minute (Fig. 3).

Length of visible jet [mm]

Figure 2. A picture of the non-thermal plasma jet.

75

70

65

60

55

1 2 3 4 5

Gas flow rate [l/min]

Figure 3. Dependence of the jet length of APPJ on

the flow rate of nitrogen.

Jostt vol 6.indd 50 7/22/10 10:09:30 PM



Contact angle [Deg.]

90

80

70

60

50

40

0 20 40 60 80 100

Distance from the orifice [mm]

Figure 4. Water contact angle of the PE film as

a function of distance from the orifice of APPJ.

The gas flow rate was 3.0 l min -1 and the exposure

time was 10 s.

Figure 4 shows the water contact angle of the

PE film as a function of distance from the orifice

of APPJ. In order to investigate the effectiveness of

PE surface modification by the APPJ, hydrophilicity

tests were carried out on the PE films before and after

the treatment. At least three drops of the test liquids

were placed on the sample and the contact angles

were measured from the profile of the drops. The

untreated sample had contact angle of 98º with water.

Whereas after the exposure to the jet, a minimum

contact angle of 39º was measured for sample placed

at a distance of 5 mm from the orifice for a time of

10 s. For the same exposure time, a contact angle of

82º was measured when the sample was placed at a

distance of 100 mm from the orifice.

Figure 5 shows the contact angle of PE sample

as a function of the exposure time in APPJ. The

1. Fridman G. (2006) Plasma Chemistry and Plasma

processes 26: 425.

2. Stoffels, E., Kieft I. and Sladek R. (2003) J. Phys. D:

Appl. Phys. 36: 2908.

3. Laroussi M. (2005) Plasma Process Polym. 2: 391.

4. Walsh J.L. and Kong M.G. (2008) Appl. Phys. Lett.

93: 111.

5. Lu X., Ye T., Cao Y., Sun S., Xiong Q., Tang Z., Xiong

Z., Hu J., Jiang Z. and Pan Y. (2008) J. Appl. Phys.

104: 053.

6. Bruggeman P. and Leys C. (2009) J. Phys. D: Appl.

Phys. 42: 053.

7.

8.

Kolb J.F., Joshi R.P., Xiao S. and Schoenbach K.H.

(2008) J. Phys. D: Appl. Phys. 41: 234.

Gonzales-Aguilar J., Moreno M. and Fulcheri L.

Contact angle [deg.]

REFERENCES

100

90

80

70

60

50

40

treatment time ranged from 1 s to 12 s. As shown in

the figure, the contact angle of the film changed from

98º for the untreated to the lowest value of 43º after

the treatment.

CONCLUSION

0 2 4 6 8 10 12

Exposure time [s]

Figure 5. Water contact angle of PE sample as

a function of the exposure time in APPJ. The

distance of the sample from the orifice was 5 mm

and the gas flow rate was 3.0 l/min.

An atmospheric pressure plasma jet has been

developed and tested. The irradiation of the jet onto

the polymer surface can improve the hydrophilicity

of the sample without any damage to the material.

The advantages of the present jet system are that the

power consumption is low and there is no need of

matching network. This plasma source can be used

for the treatment of non-planar surface and heat

sensitive materials.

(2007) J. Phys. D: Appl. Phys. 40: 2361.

9. Schutze A., Jeong J.Y, Banayan S.E, Park J., Selwyn

G.S. and Hicks R.F. (1998) IEEE Trans. Plasma Sci.

26: 1685.

10. Kunhardt E.E. (2000) IEEE Trans. Plasma Sci. 28:

189.

11. Daun Y., Hung C. and Yu Q.S. (2005) IEEE Trans.

Plasma Sci. 33: 328.

12. Borcia G., Chiper A. and Rusu I. (2006) Plasma

Sources Sci. Technol. 15: 849.

13. Benedict J., Forcke K., Yanguas-Gil A. and Keudell

von A. (2006) J. Appl. Phys. 99: 112.

14. Foster S., Mohr C. and Viol W. (2005) Surface and

Coatings Technology 200: 827.

15. Tioshifuji J., Katsumata T., Takikawa H., Sakakibara

Jostt vol 6.indd 51 7/22/10 10:09:31 PM

51

Journal of Science and Technology in the Tropics (2010) 6: 49-52


52

Journal of Science and Technology in the Tropics (2009) 6: 49-52

16.

17.

T. and Shimizu I. (2003) Surface and Coating

Tech. 171: 302.

Takemura Y., Kubota Y., Yamaguchi N. and Hara T.

(2009) IEEE Trans. Plasma Sci. 26: 1604.

Choi J., Matsuo K., Yoshida H., Namihira T., Katsuki

18.

S. and Akiyama H. (2009) Japanese J. Appl. Phys. 48:

086.

Subedi D.P., Madhup D.K., Adhikari K. and Joshi

U.M. (2008) Indian Journal of Pure and Applied

Physics 6: 540.

Jostt vol 6.indd 52 7/22/10 10:09:31 PM


Journal of Science and Technology in the Tropics (2010) 6: 53-57

Compressed natural gas (CNG) cylinder testing and data evaluation

using acoustic emission technique

Tonphong Kaewkongka 1,* , Jirapong Lim 2 and Suparerk Sirivedin 3

1Department of Physics, Faculty of Science, Chulalongkorn University, Patumwan,

Bangkok 10330, Thailand

2Department of Production Engineering, Faculty of Engineering, King Mongkut University of Technology,

North Bangkok, Bangsue, Bangkok 10800, Thailand

3King Mongkut University of Technology, North Bangkok, Bangsue, Bangkok 10800, Thailand

(*E-mail: tonphong.k@chula.ac.th)

Abstract This paper describes a method of compressed natural gas (CNG) storage cylinder testing using

acoustic emission technique. The CNG storage cylinder, which is type I-steel cylinder, was tested as

normal and predefined surface crack operating condition. Acoustic emission (AE) signals propagation and

transmissions were captured by piezoelectric transducers mounted on surface across the cylinder with and

without predefined surface crack. In the experiment, an increased step-wise condition in hydrostatic pressure

up to 400 bars was applied to the cylinder. The feature extraction and classification of AE signals from each

testing condition was applied. The results are very promising in terms of identifying the defects due to crack

propagation.

Keywords acoustic emission – CNG cylinder

INTRODUCTION

The number of vehicles using compressed natural gas

(CNG) has grown rapidly for the past decades due to

the alternate use of energy and become an important

issue in Thailand. In most cases, the nature of the

contents renders these cylinders critical to operations

and safety. There have been several severe accidents

from the explosion of CNG cylinders. Therefore, it

may lead to significant loss or catastrophic failures.

For health and safety issue, monitoring the condition

of the cylinders has received considerable attention

over the past few years due to its particular importance

of safety, environment and the economical reason [1-

3]. A reliable condition monitoring system will reduce

the failure and possible loss.

According to inspection standard, it requires that

cylinders be visually inspected externally after it

has been used for 36 months. This is to ensure that

the cylinders that have damage or deterioration will

be removed from service or repaired. The visual

inspection test needs removal of the cylinder from

the vehicle. However, there are possible risks if the

internal flaws or defects cannot be examined.

Nowadays there are many kinds of conventional

methods available for the CNG storage cylinder

detection [4-6]: visual inspection using dye penetration,

hydro-static testing, flow or pressure measurement,

ultrasonic testing. However, these methods are time

consuming due to the requirement of removing the

cylinder from the vehicle and revealing the defects of

the cylinder after it had already occurred. Recently,

acoustic emission (AE) has been extensively applied

to vessels testing during pressurization and has proven

to be a timely and economical method for structural

integrity assessment as described in the standard and

code, ISO/DIS 16148.2 [7]. A major advantage of AE

inspection is that it allows the whole structural integrity

to be tested non-intrusively with minor disruption (no

need to remove the cylinder from service, empty and

clean for inspection) in a pressurized condition. AE

can provide complete coverage of the cylinder with

pressurized test and is sensitive to active defects or

flaws presence and propagation.

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53

Journal of Science and Technology in the Tropics (2009) 5: 141-150


54

Journal of Science and Technology in the Tropics (2010) 6: 53-57

The objective of this research is to demonstrate that

a condition-based monitoring using acoustic emission

(AE) can provide not only timely detection of defects

in the cylinder but also the crack propagation so that

maintenance or replacement can be performed prior

to the loss of safety function. Therefore the use of

acoustic emission method has been proposed for

CNG type-I steel cylinder monitoring instead of the

conventional methods.

EXPERIMENTAL SETUP AND APPARATUS

Acoustic emission is a natural phenomenon of stress

wave generation and propagation spontaneously

when a material is subjected under stress [8]. Plastic

deformation and growth cracks are the primary

sources of acoustic emission in metals. The acoustic

signal can be detected by a piezoelectric transducer,

which converts the mechanical energy carried by the

elastic wave into an electrical signal.

When the defective cylinders are pressurized,

stress waves (AE) can be produced by several different

sources (e.g. secondary sources or actual propagation

of cracks). These sources can result in AE activities

generated at pressure less than, equal to or greater

than the operating pressure. The stress waves are then

propagated throughout the structure.

A systematic approach to classify the dynamic

responses of AE signatures associated with the

CNG type I steel cylinder operating condition was

performed in this study (Fig. 1). The AE testing is

usually carried out during a controlled pressurization

of a cylinder. Conventional AE parameter, AE rms is

used to identify the presence of acoustic emission

activities produced when microscopic cracks occur.

The AE rms can be calculated using the formula below

[9]:

where v ( t)

is the electrical voltage signal obtained

from piezoelectric transducer, t o is the initial time,

T is the period of the AE activity.

Figure 1. A system of acoustic emission testing.

Figure 2. Set up of CNG cylinder testing with four installed AE

sensors.

Jostt vol 6.indd 54 7/22/10 10:09:35 PM



The acoustic emission sensors, model AE-

SS1 (Holroyd Instruments, UK) which were made

of piezoceramic elements, were mounted on the

circumference along the surface of the cylinder

(Fig. 2). The acoustic emission sensors of 100 kHz

resonance frequency responded well with the material

degradation and microscopic crack initiation and

growth. The captured acoustic emission signals were

then conditioned and amplified with 60 dB-gain signal

conditioning unit. The PCI-1714UL was used for

A/D conversion and data logger. In the experiments,

a normal operating cylinder was tested with four AE

sensors by means of mechanical coupling (Fig. 2).

The cylinder used in this experiment had a 60 litre

capacity. It was made of a steel sheet with a diameter

of 300 mm, length of 990 mm and thickness of 8

mm.

To perform hydrostatic test, the cylinder was

placed in a chamber which could facilitate a high

pressure testing (Fig. 3). For an abnormal operating

condition cylinder, a simulated surface crack was

performed on a good cylinder. The simulated

(artificial) crack (Fig. 4) was made using a grinder to

generate a surface defect with length of 30 mm (along

the length of cylinder), width of 3 mm and depth of

2 mm.

To eliminate background noise, the threshold of

AE signal was set to 39 dB AE . Evidently, the threshold

level affects the value of the AE parameters. A typical

Figure 3. Installation of CNG cylinder in a

high-pressure hydrostatic test chamber.


example is the event duration. By definition, it is the

time that the AE event is above the threshold. A step

wise increase in hydrostatic pressure was performed

up to 400 bars with the testing cylinder. During the test,

a pressure sensor was attached with the hydro-testing

machine as for load reference for data acquisition.

EXPERIMENTAL RESULTS AND

DISCUSSION

To verify the proposed approach, the experiment on

the normal operating cylinder was performed using

hydrostatic test. A hydrostatic test was applied on the

testing cylinder with increased pressure up to 300

bars. The AE signals captured from all four sensors

Figure 4. Artificial surface crack generated

at the side wall of the cylinder.

Figure 5. AE signals versus time (data point)

from a normal CNG cylinder.

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Journal of Science and Technology in the Tropics (2010) 6: 53-57


56

Journal of Science and Technology in the Tropics (2010) 6: 53-57

attached on the good or normal operating condition

cylinder yielded a calm condition in a random

background noise fashion (Fig. 5).

In the experiment, an artificial surface crack

(300mm length, 2 mm width) cylinder was tested with

the applied pressure up to 300 bars. The experimental

results are shown in Figure 6. It illustrates that the

time domain signals of the artificial defective wall

cylinder gave rise to the abrupt change in higher

amplitude to all four AE sensors.

Ultimately, the pressure was increased up to

400 bars which resulted in the crack of the artificial

defect of the wall. At this state, the water was leaking

out due to the open surface of the previous artificial

crack because it could not sustain the increased the

internally applied load any further. This resulted in

the AE signals as shown in Figure 7.

The AE rms values calculated from the time domain

signal are shown in Figure 8. For the normal cylinder,

it gave the lowest signal energy around 15.8 mV.

This is because there was only background level of

the hydrostatic test that was present. It illustrates

that the defective cylinder condition yielded the

highest value about 211 mV. Its value was about 13.3

times greater than the one from normal operating

condition. This is due to the microscopic crack growth

as the primary source and the turbulent flow at the

surface of the crack as secondary source. Whereas

the leakage cylinder shows that the AE rms values were

relatively greater than the normal condition at about

20.8 mV (greater by 5 mV).

The methodology described in this paper has

been shown to work well for monitoring CNG

cylinder operating conditions. The method involves

calculation of AE rms values as a mean to identify the

abnormal operating conditions of the CNG cylinder

together with hydrostatic test. Overall the proposed

method can provide timely detection of the presence

of crack and leakage of the CNG cylinder and is

efficient to implement.

1. ASME (2000) Boiler and Pressure Vessel Section XI,

Appendix A, Article A-3000.

2. ASTM E2191-08 (2006) Standard Practice for

Examination of Gas-Filled Filament – Wound

Composite [3] Pressure Vessels Using Acoustic

Emission.

REFERENCES

Figure 6. AE signals versus time (data point) from a

defective CNG cylinder.

Figure 7. AE signals versus time (data point) from a

leaked CNG cylinder.

Figure 8. Results of AE rms from different operating

conditions.

3. ASTM E1419-02b (2002) Standard Test Method for

Examination of Seamless, Gas-Filled, Pressure Vessels

Using Acoustic Emission.

4. Connolly M.P. and Han Dinh (1996) Fleet Inspection

of Compressed Natural Gas Cylinders for Natural Gas

Vehicles Using Source Location Acoustic Monitoring.

Jostt vol 6.indd 56 7/22/10 10:09:50 PM


SAE Technical Paper Series No. 961174.

5. Webster C. (2007) Development of Non-Destructive

Evaluation (NDE) Techniques for CNG Fuel Tanks.

Report for Transportation Development Centre of

Transport Canada.

6. Hudak S.J., Jr. (1991) Assuring the Safety of Natural

Gas Vehicles. Technology Today Magazine, Sept.

1991.

7. ISO/DIS 16148.2 (2006) Gas cylinders – Refillable

seamless steel gas cylinder – Acoustic emission

examination for periodic inspection.

8. Pollock A.A. (1973) Acoustic Emission 2 – Acoustic

emission amplitudes. Journal of Non-Destructive

Testing10: 264-269.

9. Scruby C.B. (1987) An introduction to acoustic

emission. Journal of Physics Instrument Science and

Technology 20: 946-953.

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Journal of Science and Technology in the Tropics (2010) 6: 59-65

Prompt gamma neutron activation analysis (PGNAA) of hydrocarbons:

A Monte Carlo study with GEANT4

Lam YiHua1 , Wong Chiow San1 * and Kurunathan Ratnavelu2 1Plasma Research Laboratory, Department of Physics, Faculty of Science, University of Malaya,

50603 Kuala Lumpur, Malaysia

2Institute of Mathematical Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

*Corresponding author

Abstract In the present work, the results of prompt gamma (γ) neutron activation analysis (PGNAA)

calculated by the open source GEANT4 Monte Carlo toolkit were compared with the commercial simulation

package MCNP. Both the Monte Carlo simulation packages utilize the phenomenological nuclear reactions

to yield γ rays which are coupled with incident neutrons after these reactions. A virtual experimental setup

with a 4π solid-angle γ detection was instantiated in the simulations to collect all prompt γ rays from the

tested hydrocarbon phantoms. The comparative results present good agreement in getting the characteristic

γ energy spectra of hydrocarbon materials.

Keywords GEANT4 – MCNP – Monte Carlo simulations – prompt gamma neutron activation

INTRODUCTION

A material with its specific chemical composition and

density can be detected or differentiated from among

other types of materials, which have nearly the same

chemical composition but have not complied with the

same densities, by using prompt gamma (γ) neutron

activation analysis (PGNAA) method. It has been

used in detecting explosive hydrocarbon material [1].

In addition, certain element in a bulk material or in a

biological tissue can be determined via PGNAA, e.g.

biomedical protein detection [2].

PGNAA method relies on neutron-nuclear

interactions, that is, inelastic scattering (n, n′γ), (n, pγ)

and radiative capture (n,γ), to yield γ rays. Although

1/v law [3] (v is the incident neutron velocity) is not

applicable to every element, isotope and energy range

[4], it is still acceptable to be applied in low Z element

and in thermal energy range [5]. This law infers that

the probability of neutron radiative capture depends

on how long the period of time a neutron spends in

the region surrounding the nucleus. The longer period

of time it spends, the higher probability of neutron

radiative capture will occur. Hence, when a low

kinetic energy incident neutron, e.g. thermal neutron

(~0.025eV) or epithermal neutron (0.1-1eV), enters the

narrow separation virtual energy levels in compound

nucleus or interacts with compound nucleus [6], the

neutron radiative capture cross sections will increase.

These low energy interactions raise the possibility of

producing stable isotopes and the coupled γ ray, e.g.

1 H(n, γ) 2 H in which 2 H is a stable isotope and γ ray will

be radiated. Besides, different materials composed by

different chemical compositions (including different

isotopes) and densities will have different radiative

capture cross sections. By radiating low energy

neutrons with certain range of energy distribution on

different materials, the produced γ ray energy spectra

will be different. Hence, the prompt γ ray energy

spectra become an identifying characteristic of the

material.

Other than experimental measurements, PGNAA

can be simulated by using various types of Monte

Carlo computational simulation packages, such as

GEANT4 [7,8], MCNP [9] and FLUKA [10,11].

Most of these packages are written in structural

programming language (C and FORTRAN). GEANT

4 has been written in object-oriented programming

(OOP) architecture (C++), developed and maintained

by many laboratories [7,8]. Conceptually, OOP has

its distinctive feature in treating particle as a single

object which may have encapsulated its own data

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60

Journal of Science and Technology in the Tropics (2010) 6: 59-65

structure (characteristic data and methods) and

respective particle transport physics model. The OOP

methodology (the way in composing program) can

resemble the physical occurrence, e.g. a neutron,

which is treated as an object in the simulation,

may collide and be captured by a thermal nucleus

(the second object) to yield γ ray (the third object);

alternatively, the neutron may undergo a beta decay

process to produce a proton (the second object), an

electron (the third object) and an anti-neutrino (the

fourth object). The created electron may produce

γ ray (the fifth object) during Bremsstrahlung

process. During radiative decay, annihilation and

radiative capture, the interacting particles will be

deleted from the computer memory after yielding

respective secondary particle(s). In this way, the OOP

computational method resembles the physics process

as the active interacting particle, e.g. electron, positron

and neutron, will disappear after its respective physics

process, e.g. annihilation or radiative capture.

In this work, the incident neutrons are assumed

to be thermalized and their energies are in accord

with Maxwell-Boltzmann (MB) distribution, and the

incident energy of highest probability is 0.025 eV. The

prompt γ energy spectra of rubber, which are calculated

by GEANT4 by means of either inducing thermalized

incident neutrons or inducing monoenergetic incident

neutrons on target, is compared with the results of

prompt γ energy spectra induced by monoenergetic

incident neutrons, which was generated by the MCNP

[12].

SIMULATION OF PGNAA

Physics list and incident random energy

generator

The calculations of the current PGNAA Monte Carlo

simulation were processed by GEANT4.8.2.p01,

Detector


Construction


Virtual


Experimenta

l
Setup


Registere

Registered

d


Materials Materials


G4NDL3.10,
etc


GEANT4


Graphical


Visualization


General
Particle
Source



(Maxwell­Boltzman
Distribution)


CLHEP
random
number
generator


Figure 1. Schematic organization of PGNAA simulation program.

whereas the evaluated neutron cross section input

data was by G4NDL3.10, a recompiled data of a few

evaluated nuclear data (including ENDF-B VI, JENDL,

FENDL, CENDL, Brond, EFF, JEF, MENDL) [7].

Besides, data sets such as G4EMLOW2.3 were used as

part of the input data to facilitate the calculation of the

γ propagation handled by electromagnetic processes

(Low Energy Electromagnetic). These data files were

input via respective physics process classes. Figure 1

shows the schematic flow of the application program

of GEANT4.

Neutron Elastic, Inelastic, Capture and Fission

handled by the neutron high precision (HP) model

[7], [13] are considered in this PGNAA simulation.

These four low energy neutron physics models are

able to describe high precision final state production

which is based on the G4NDL3.10. It covers the

neutron scattering energy range from thermal energy

up to 20 MeV. In addition, Doppler broadening and

thermal motions of target nucleus are covered in the

final state generator. Both processes are done on-thefly

by the neutron HP models and their associated

cross sections, G4NDL. Although fission is quite

rare to happen, it is included in the simulation as a

precaution in the PGNAA simulation. These four

processes are considered as post-step reactions and

one of them is selected via random number selection

by means of comparing the mean free path. Before

the selection of physics processes, an element will

be selected among elements composing a material

and an isotope will be selected from among isotopes

which are defined for that element. These two levels

of selection (of element and isotope) are calculated

via random number selection and comparison of

the respective cross sections in GEANT4 kernel

(class G4HadronicProcess). However, inside the

class G4HadronicProcess, the original method

G4HadronicProcess::ChooseAandZ assumes isotope

Stepping


Action


ASCII


Output
Results


(γ
energy)


Analysis


Manager


Output
Results



(γ
energy)


AIDA
Interface



Grace5.1.21


(Data
Analysis)


JAS3


(Data
Analysis)


Jostt vol 6.indd 60 7/22/10 10:09:53 PM


abundance values by retrieving the default values in an

instantiated object of G4StableIsotope. These isotope

abundance values may not suit the user need. Hence,

the method G4HadronicProcess:ChooseAandZ

had been altered to suit the user defined isotope

abundance values [14].

Besides, the simulations have been separately run

with two sets of electromagnetic physics models: (1)

Standard Electromagnetic physics model; and (2)

Low Energy Electromagnetic physics models. Both

electromagnetic physics models consist of e - e + pair

production, photoelectric, γ conversion and Compton

scattering. Extra physics models have been attached

to e - and e + : (1) Multiple Scattering; (2) Low Energy

Ionization and Low Energy Bremsstrahlung; or

Standard Ionization and Standard Bremsstrahlung;

whereas Low Energy Rayleigh has been attached

to γ. However, we find that both sets of physics

model – Standard Electromagnetic and Low Energy

Electromagnetic produce the same outcomes.

Therefore, only one set of results is shown in this

paper. Furthermore, the registered processes for

proton and ions (deuteron, triton, 3 He, alpha, generic

ion) are Multiple Scattering, Low Energy Ionization

and respective hadronic routines e.g. low energy

proton/deuteron/triton/alpha inelastic and elastic

processes.

We assume the process of neutron thermalization

will eventually produce neutrons with energy

distribution described by Maxwell-Boltzman (MB)

distribution,

(1)

Instead of integrating the distribution function to

yield the randomized incident neutron energies, the

function can be treated in the form of a histogram

with 10000 or more bins. Then the histogram can

be normalized to its maximum value. The output

of this function f(E) can be cumulated as a discrete

cumulative distribution [15]. Even random numbers

between 0 and 1, which are generated via a Class

Library for High Energy Physics (CLHEP-2.0.3.1),

are distributed on the ∑f(E)-axis. The corresponding

x-axis values are the MB randomized incident

neutron energies. The MB distribution code has

been embedded into a new class inherited from

the class G4GeneralParticleSource. The highest


probability energy of the random distribution is

0.025eV within the energy range of 0.001eV < E <

0.050eV.

Virtual experimental setup

The listed isotopes in Table 1 and materials in

Table 2 were constructed in a user defined detector

construction class and registered into the GEANT4

kernel. Figure 2 shows the experimental setup, which

consisted of two spherical volumes (phantoms), i.e.

the internally occluded sphere (black, with radius

0.62 cm) and the exterior occluding hollow sphere

(grey, with radius 1.05 cm). The setup was similar

to Nunes et al. [12] and Sohrabpour et al. [16]. The

hollow sphere and the inner spheres could be either

set as the same material or set as different materials.

The total physical volume of both spheres was 1

cm 3 . Therrmalized neutrons would be directed in

Table 1. List of registered isotopes.

Registered isotopes Natural abundance (%)

1 H 99.985

2 H 0.015

12 C 98.80

14 C 1.20

16 O 99.757

17 O 0.038

18 O 0.205

14 N 99.640

15 N 0.360

Table 2. List of registered materials.

Registered Chemical Density

materials composition (g/cm3 Temperature

) (K)

C4

Rubber

C H O N 4 6 6 6

C H 5 8

1.83

0.94

273.15

273.15

Figure 2. A typical run sample (snapshot) with

prompt γ rays.

Jostt vol 6.indd 61 7/22/10 10:09:55 PM


61

Journal of Science and Technology in the Tropics (2010) 6: 59-65


62

Journal of Science and Technology in the Tropics (2010) 6: 59-65

parallel to the both spheres. The source of neutrons

was confined in a plane square (2.5 × 2.5) cm 2 , and

located 5 cm (arbitrary) from the spheres. As long

as the yield γ ray of inelastic (n, n'γ) or capture

(n,γ) reaction propagated from spherical volumes to

atmosphere in 4π directions, energy of the prompt γ

ray would be cumulated for further analysis. There

was no variance reduction applied on the analysis.

During the simulation, random neutron incident

energy and energy of prompt γ ray results were kept

in ASCII and Abstract Interfaces for Data Analysis

(AIDA) 9170 format files for further analysis which

was respectively based on Grace and Java Analysis

Studio (JAS3).

Figure 2 shows a typical run of the simulation.

Both spherical volumes (inner and outer) were set as

explosive C4, and they were placed in the ambience of

atmospheric gas (70% N 2 , 30 % O 2 ), alternatively, the

ambience could be set as vacuum too. The spherical

volumes were exposed to 100 incident neutrons with

MB energy distribution. Light grey lines represent

the neutron tracks (incident neutrons or scattered

neutrons). This typical run produced two γ tracks

(grey lines) as labeled in Figure 2. The chemical

composition of C4 is given in Table 2.

RESULTS AND DISCUSSION

In this PGNAA simulation, γ energy spectra of

a typical plastic explosive (C4), rubber, and C4

occluded by rubber were calculated and compared.

Figures 3-5 show the comparison of γ energy spectra

produced by GEANT4.8.2.p01 and MCNP (analyzed

by Nunes et al.) [12]. The γ energy spectra represent

the interactions between the thermal neutrons and

the nucleus of the constituent elements of C4 and

rubber, and represent also γ interactions in the sample

material itself. The ratio of non-neutron-capture

(i.e. ionization, Bremsstrahlung, transportation,

annihilation, radioactive decay and multiplescattering)

processes, which are related to γ yield, to

neutron capture process is ~ 0.08. It may be infered

that thermal neutron capture process plays a significant

role in γ production. The identity of the constituents

of the particular material can then be adduced. There

are three configurations of virtual experimental

setup: (1) both inner and outer spherical phantoms are

placed in atmospheric environment and are irradiated

by MB distributed energy neutrons (the upper most

histogram); (2) both spherical phantoms are located

in vacuum and are irradiated by MB distributed

Figure 3. Prompt γ energy spectra of C4 generated by GEANT4.8.2.p01 compared with MCNP.

Jostt vol 6.indd 62 7/22/10 10:10:09 PM


energy neutrons (the second upper histogram); and

(3) both spherical phantoms are situated in vacuum

and are irradiated by monoenegetic neutrons (the

second lower histogram); with (4) γ energy spectra

calculated by MCNP.

In Figure 3, Nunes et al.’s results show that

hydrogen contributed to the peak of prompt γ ray (2.2

MeV), carbon (4.9 MeV) and nitrogen (10.8 MeV).

Their results illustrate that the explosive spectra have

significant γ energy peaks which are the signatures

for C4 from 1 to 12 MeV (with 100 histogram bins

ranges from 1 – 12 MeV).

Obviously, a few major peaks are consistently

observed on the corresponding γ energy spectra

produced by GEANT4.8.2.p01 (Figure 3). Three of

the major peaks (~14600 counts of 2.2 MeV, ~130

counts of 4.9 MeV, ~720 counts of 10.8 MeV) match

with γ energy spectra produced by MCNP as stated

above. Other than the three major peaks, γ energy

spectra produced by carbon from inelastic interactions

(marked with *) and from s-wave captures include

1.85 MeV, 3.68 MeV, and *2.08 MeV. In addition,

nitrogen contributes *2.52 MeV, *2.90 MeV, *3.88

MeV, *4.50 MeV, 5.27 MeV, 5.30 MeV, *5.53 MeV,

*5.56 MeV, 6.32 MeV, 7.30 MeV, 8.31 MeV, 9.15

MeV, 9.93 MeV; whereas oxygen generates γ energy

spectrum with 3.05 MeV. All the γ energy spectra

above are in agreement with those published in

Ref. [19]. It is clear that γ energy spectra calculated

by GEANT4.8.2.p01 are discrete at 2.2 MeV and

above, whereas γ energy spectra below 2.2 MeV are

continuous. This is due to the various electromagnetic

processes, for instance, Bremsstrahlung, occur in this

energy range.

In Figure 4, a few extra γ energy spectra peaks

are recorded at 4.50 MeV, 5.27 MeV, 5.30 MeV, 5.53

MeV, 5.56 MeV, 6.32 MeV, 7.30 MeV, 9.15 MeV,

10.8 MeV in the top histogram compared to the other

two histograms below. These extra energy spectra

are similar with nitrogen peaks in Figure 3. These

background energy spectra may be contributed by the

atmospheric gas as the chemical composition setting

of rubber (C 5 H 8 ) does not contain nitrogen, besides,

nitrogen peaks do not appear in vacuum configuration

of these two experimental setups.

When C4 is occluded by rubber, the original

rubber’s prompt γ energy spectra will mix with

characteristic γ energy spectra from C4. This mixture

is clearly shown in Figure 5. High peak of the nitrogen

(10.8 MeV) and many extra γ energy spectra produced

Figure 4. Prompt γ energy spectra of Rubber (C5H8) generated by GEANT4.8.2.p01 compared with

MCNP.

Jostt vol 6.indd 63 7/22/10 10:10:20 PM


63

Journal of Science and Technology in the Tropics (2010) 6: 59-65


64

Journal of Science and Technology in the Tropics (2010) 6: 59-65

by nitrogen indicates that the occluded material has

a high concentration of nitrogen. This indication

not only differentiates rubber with C4 from rubber

without C4, but also depicts the ratio of nitrogen in

each phantom in order to determine the presence of

plastic explosives [20].

Furthermore, Figure 3 and Figure 5 show that

incident neutrons with MB distribution energy will

only trigger slightly more extra γ energy spectra

compared to mono energetic neutrons. This outcome

is plausible as: (1) more types of elements in the

chemical composition and MB neutron energy

(more random energy) will give rise to more

possibilities of inelastic scattering; (2) although

the more random of the incident energy, the more

transitions incurred by inelastic scattering and

capture processes are expected, the difference

of randomized neutron energies is small. Hence,

small range of randomized neutron energy does not

impact much to the prompt γ energy spectra.

Figure 4 does not show an obvious difference of γ

energy spectra between MB and mono energetic.

Obviously, there are not many types of element in

the case of neutron-rubber scattering. The targets

of these simulations are in static form, Doppler

broadening does not affect the outcome of γ

energy spectra. Overall, the γ energy spectra

calculated by GEANT4.8.2.p01 shows good

agreement with MCNP in terms of the ratio of γ

energy spectra counts in histograms.

CONCLUSION

The present study on PGNAA computational

simulation which focuses on the comparison of

the corresponding characteristic γ rays is able to

show the differences among explosive and nonexplosive

occluded materials. Besides, we

demonstrate that γ rays generated from s-wave

neutron captured are not the only energy

spectrum to show the differences, non s-wave

neutron capture can also yield γ rays, which are

able to provide some significant energy peaks

for identifying hydrocarbon materials, e.g.

N (5.53 MeV) and N (5.56 MeV) which have

almost the same count as N (10.83 MeV) and

higher than C (4.95 MeV). Although the incident

neutron energies are randomly generated according

to Maxwell-Boltzmann distribution in mode 0.025

eV, the prompt γ spectra can still be able to show

Figure 5. Prompt γ energy spectrum of C4 hidden by Rubber generated by GEANT4.8.2.p01

compared with MCNP.

Jostt vol 6.indd 64 7/22/10 10:10:31 PM


the differences of γ energy spectrum for identifying

some hydrocarbon materials. The attempt of making

use of random incident neutron energy instead of

mono energetic neutron energy has paved the way

to imitate the physical condition in computational

simulation. For the time being, we consider GEANT4

should be a good alternative and candidate

for PGNAA simulation.

1. Shea P. M., Gozani T. and Bozorgmanesh M. (1990)

Nucl. Instrum. Methods Phys. Res. A, 299: 444.

2. Nagai T., Fujii I., Muto H. and Inouye T. (1969) J.

Nucl. Med. 10: 192.

3. Bethe H. A. (1935) Phys. Rev. 47: 747.

4. Rasetti F., Segrè E., Fink G., Dunning J. R., and

Pegram G. B. (1936) Phys. Rev. 49: 104.

5. Bethe H. A. (1936) Rev. Mod. Phys. 9: 69.

6. Soete D. De, Gijbels R. and Hoste J. (1972) Neutron

Activation Analysis. Wiley-Interscience, England.

7. Agostinelli S. et al (2003) Nucl. Instrum. Methods

Phys. Res. A 506: 250.

8. Allison J. et al (2006) IEEE Trans. Nucl. Sci. 53:

270.

9. Breismeister J.F. (1997) MCNP: a General Monte

Carlo N-Particle Transport Code, Version 4B, LA-

12625-M. Los Alamos National Lab., New Mexico.

10. Fasso’ A., Ferrari A., Ranft J. and Sala P. R. (2005)

FLUKA: a Multi-Particle Transport Code, CERN

2005-10, INFN/TC_05/11, SLAC-R-773.

11. Fasso’ A., Ferrari A., Roesler S., Sala P. R., Battistoni

G., Cerutti F., Gadioli E., Garzelli M. V., Ballarini F.,

Ottolenghi A., Empl A. and Ranft J, (2003) The Physics

Models of FLUKA: Status and Recent Developments,

Computing in High Energy and Nuclear Physics

REFERENCES

Acknowledements – This work was supported in part by

the University of Malaya Fundamental Research Grant

(FP010/2004A). The author would like to thank Hans-

Peter Wellisch (hadronic process), Mark Donszelmann

and Andreas Pfeiffer (data analysis), Vladimir

Ivantchenko and Gabriele Cosmo (system installation),

John Allison and Fan Lei (particle track visualization)

for their suggestions and support.

12.

2003 Conference (CHEP2003), La Jolla, California,

USA (paper MOMT005) eConf C0303241 (2003),

arXiv:hep-ph/0306267.

Nunes W. V., Silva A. X., Crispim V. R. and Schirru R.

(2002) Appl. Radiat. Isot. 56: 937.

13. Wellisch J. P. (2005) April 17-21 2005 The Neutron_

HP Neutron Transport Code, Monte Carlo 2005

Topical Meeting ISBN: 0-89448-695-0, Chattanooga,

Tennessee, USA.

14. [Online] Available: http://geant4-hn.slac.stanford.edu:

5090/HyperNews/public/get/hadronprocess/672/1/3.

html.

15. Garcia A. L. (2000) Numerical Methods for Physics,

2nd ed. Prentice Hall, New Jersey.

16. Sohrabpour M., Shahriari M., Zarifian V. and

Moghadam K. K. (1999) Appl. Radiat. Isot. 50: 805.

17. [Online] Available: http://aida.freehep.org/index.

thtml.

18. Zhou ChunMei July 2000 Thermal Neutron Capture

Data for A=1-25, IAEA, INDC(CPR)-051 [Online]

Available: http://www-nds.iaea.org/reports-new/indcreports/indc-cpr.

19. Vourvopoulos G and Womble P C 2001 Pulsed Fast/

Thermal Neutron Analysis: a Technique for Explosives

Detection, TALANTA 54: 459.

Jostt vol 6.indd 65 7/22/10 10:10:32 PM

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Journal of Science and Technology in the Tropics (2010) 6: 59-65


66

Jostt vol 6.indd 66 7/22/10 10:10:32 PM


Volume 6 Number 1 Jun 2010

Editorial

Salleh Mohd. Nor and Mohinder Singh 3

Comparison of water and gastrografin as an oral contrast medium for

abdominopelvis computed tomography

K. C. Kanaga, S. E. Laila, P. Y. Teh and J. Shukriah 5

Effect of colchicine on tissue culture derived plants of Zingiber officinale Rosc.

and Zingiber officinale var. rubrum Theilade

Lindayani, K. Norzulaani, H. Ibrahim and Noorsaadah Abd Rahman 11

Diversity and abundance of Dacinae fruit flies (Insecta: Diptera: Tephritidae) in Chini 2,

Runchang and Sungai Bebar, Pahang, Peninsular Malaysia

Hoi Sen Yong, Y. F. Ng and Phaik Eem Lim 17

The mammal fauna of Pulau Singa Besar, Langkawi, Kedah, Peninsular Malaysia

Lim Boo Liat, Norhayati Ahmad, O. Noor Alif Wira, Shahrul Anuar Md. Sah 23

Antiproliferative and antioxidant properties of leaf extracts of Pereskia bleo (Cactaceae) and

their ability to limit natural and oxidant-induced apoptotic cell death

Hui Meng Er, Hong Lim Lee, Ammu Kutty Radhakrishnan 31

Abdominal colour polymorphism in female Asian Golden Web Spider Nephila antipodiana

(Araneae: Nephilidae)

Yong Hoi Sen, Rosli Hashim, Daicus Belabut and Lim Phaik Eem 39

Scaled down operation of the United Nations University/International Centre for

Theoretical Physics Plasma Focus Facility (UNU/ICTP PFF) as an extreme ultraviolet source

Rattachat Mongkolnavin, Prajya Tangitsomboon and Chiow San Wong 43

An atmospheric pressure non-thermal plasma jet in nitrogen for surface modification of polyethylene

D. P. Subedi, R. B. Tyata, A. Shrestha, D. Baral, D. K. Madhup and C. S. Wong 49

Compressed natural gas (CNG) cylinder testing and data evaluation using

acoustic emission technique

Tonphong Kaewkongka, Jirapong Lim and Suparerk Sirivedin 53

Prompt gamma neutron activation analysis (PGNAA) of hydrocarbons:

A Monte Carlo study with GEANT4

Lam YiHua, Wong Chiow San and Kurunathan Ratnavelu 59

Jostt vol 6.indd 1 7/22/10 10:08:24 PM


JOURNAL OF SCIENCE AND TECHNOLOGY IN THE TROPICS

INSTRUCTIONS TO CONTRIBUTORS

JOSTT is a multi-disciplinary journal. It publishes original

research articles and reviews on all aspects of science

and technology relating to the tropics. All manuscripts

are reviewed by at least two referees, and the editorial

decision is based on their evaluations.

Manuscripts are considered on the understanding that

their contents have not been previously published, and

they are not being considered for publication elsewhere.

The authors are presumed to have obtained approval

from the responsible authorities, and agreement from all

parties involved, for the work to be published.

Submission of a manuscript to JOSTT carries with

it the assignment of rights to publish the work. Upon

publication, the Publishers (COSTAM and ASM) retain the

copyright of the paper.

Manuscript preparation

Manuscripts must be in English, normally not exceeding

3500 words. Type double spaced, using MS Word, on one

side only of A4 size with at least 2.5 cm margins all round.

Number the pages consecutively and arrange the items

in the following order: title page, abstract, key words, text,

acknowledgements, references, tables, figure legends.

Title page

Include (i) title, (ii) names, affiliations and addresses of all

authors, (iii) running title not exceeding five words, and

(iv) email of corresponding author.

Abstract and key words

The abstract, not more than 250 words, should be

concise and informative of the contents and conclusions

of the work. A list of not more than five key words must

immediately follow the abstract.

Text

Original research articles should be organized as follows:

Introduction, Materials and Methods, Results, Discussion,

Acknowledgement, References. The International

System of Units (SI) should be used. Scientific names and

mathematical parameters should be in italics.

References

References should be cited in the text as numbers

enclosed with square [ ] brackets. The use of names in

the text is discouraged. In the reference section, the

following examples should be followed:

1. Yong H.S., Dhaliwal S.S. and Teh K.L. (1989)

A female Norway rat, Rattus norvegicus,

with XO sex chromosome constitution.

Naturwissenschaften 76: 387-388.

2. Beveridge W.I.B. (1961) The Art of Scientific

Investigation. Mercury Book, London.

3. Berryman A.A. (1987) The theory and

classification of outbreaks. In Barbosa P. and

Schultz J.C. (eds.) Insect outbreaks pp. 3-30.

Academic Press, San Diego.

Tables

Tables should be typed on separate sheets with short,

informative captions, double spacing, numbered

consecutively with Arabic numerals, and do not contain

any vertical lines. A table should be set up to fit into the

text area of at most the entire page of the Journal.

Illustrations

Black-and-white figures (line drawings, graphs and

photographs) must be suitable for high-quality

reproduction. They must be no bigger than the printed

page, kept to a minimum, and numbered consecutively

with Arabic numerals. Legends to figures must be typed

on a separate sheet. Colour illustrations can only be

included at the author’s expense.

Proofs and reprints

Authors will receive proofs of their papers before

publication. Order for reprints must be made when

returning the proofs.

Submission

Manuscripts (including all figures but not original

artwork), together with a CD version of the text, should

be submitted to:

The Editorial Office

Journal of Science and Technology

in the Tropics

Academy of Sciences Malaysia

902-4 Jalan Tun Ismail

50480 Kuala Lumpur, Malaysia

E-mail: jostt@akademisains.gov.my

JOSTT is listed in Scopus

Jostt vol 6.indd 2 7/22/10 10:08:24 PM


Journal of Science and Technology in the Tropics (2010) 6: 3

Editorial

PUBLISH OR PERISH

When one chooses a scientific career, it becomes a norm to publish. One has to disseminate one’s

findings and one’s work. It is part of the challenge of being a scientist and it is expected of a scientist.

Some institutions set target for scientists to publish at least two scientific papers in a refereed journal

a year. Such targets are good as there is a propensity for scientists in general not to publish.

There are many forms of publications. The usual is a scientific journal and most professions or

institutions have their own dedicated journals to provide a conduit for their scientific staff to publish.

However, the growing concerns on the environment and the destruction of forests have been used

as an argument to choose electronic publications and the widespread use of the internet allows this.

However, among the more senior scientists, the preference is still the written media. Publishing in

peer-reviewed journals should be encouraged as this will provide a more international audience. The

impact factor of the journal should also be taken into consideration. The number of times that an

article is cited or the citation index is also an indicator of the readership of the article.

The other issue often debated is proceedings of seminars and conferences. Conferences, workshops

and seminars provide excellent channels for the dissemination of research findings and ideas, and

proceedings of such meetings are critically important to record the presentations that would be lost

otherwise.

There is an increasing propensity for joint authorship of papers. While this is inevitable considering

that research is seldom undertaken by an individual but rather by a team, the tendency for the head of

the organization to be the main author should not be encouraged unless he or she was really the main

researcher involved in the project. The main scientist that undertook the major part of the research

and who wrote the article must be the main author.

Some disciplines are more amenable to publications. For example, in taxonomic research it is

much easier to publish when the scientist has completed the study of a certain species of plant or

animal or microorganism. However, in some other disciplines, it is much more difficult and time

consuming. Nonetheless, the key to good research is innovation and such innovative discoveries or

ideas should be published.

Finally there is the fundamental issue of scientific writing. Many scientists have never been taught

the art or science of scientific writing. It is proposed that every young scientist undergo a course on

scientific writing early in their career as this will give them confidence and provide them a good

foundation to publish their findings later.

Dr. Salleh Mohd. Nor and Dr. Mohinder Singh

Co-Chairman, Editorial Board

Jostt vol 6.indd 3 7/22/10 10:08:25 PM


Jostt vol 6.indd 4 7/22/10 10:08:25 PM


Journal of Science and Technology in the Tropics (2010) 6: 5-9

Comparison of water and gastrografin as an oral contrast medium for

abdominopelvis computed tomography

K. C. Kanaga 1 , S. E. Laila1, P. Y. Teh 1 and J. Shukriah 2

1 Diagnostic Imaging and Radiotherapy Programme, Faculty of Allied Health Sciences,

Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd Aziz, 50300 Kuala Lumpur, Malaysia

(Email: kanagakkc@yahoo.com)

2 Diagnostic Imaging Department, Kuala Lumpur Hospital, Jalan Pahang, 50300 Kuala Lumpur, Malaysia

Received 25-01-2010; accepted 06-04-2010

Abstract The aim of this study was to determine if water was comparable to gastrografin as an oral contrast

medium in defining anatomic details of abdominopelvis computerized tomography. A total of 98 patients referred

to the Diagnostic Imaging Department, Hospitak Kuala Lumpur from January to April 2009 for abdominopelvis

CT with intravenous contrast enhancement were studied using Siemens Somatom Sensation 16 scanner. The

results showed that water, with superior distension and delineation capabilities of the gaster and bowel, was

comparable to gastrografin as an oral contrast medium.

Keywords gastrografin – water – contrast medium – quantitative – qualitative

INTRODUCTION

Multidetector computed tomography (CT), with high

speed multiplanar imaging has led to a paradigm

shift in abdominopelvis imaging. CT is also capable

of producing thinner slices within short scan time

and fast intravenous infusion of contrast material

[1]. With this new technique, the use of highattenuation

positive contrast medium to outline

the stomach and small bowel may degrade image

quality especially in procedures requiring maximum

intensity reconstruction [2]. Besides, adequate mural

enhancement for diagnosis of abnormalities may

be absent due to the almost non-existent difference

between enhanced bowel wall and high attenuation

intraluminal content [3]. An oral contrast medium

should have a high distension capability that delivers

optimal differentiation between lumen and bowel

wall. Excellent mixing of contrast medium and

the contents of gastro-intestinal tract may lead to

consistent pacification and accurate interpretation of

the images [4].

Neutral contrast agent produces improved image

quality and facilitates the diagnosis of bowel wall

abnormalities. It has been proven to be effective in the

diagnosis of Crohn’s disease [5], neoplasms [6] and

bowel-ischemia [7]. Distension and delineation of the

stomach and duodenum in imaging of pancreatic and

biliary disease has been demonstrated using neutral

contrast [8]. The most widely used neutral contrast

agent is water as it has shown excellent result in the

upper gastrointestinal tract; however its clinical use

for the distal parts of the small bowel is limited due to

its rapid absorption [9].

Studies have been done to investigate a negative

contrast agent that can be used routinely for all

types of CT abdominopelvis as an alternative for the

positive oral contrast agent but the results obtained

have been inconclusive [10, 11].

This study was designed to determine if water

was comparable to gastrografin as an oral contrast

medium in defining anatomic details. The hypothesis

of our study was water would be as effective or even

better then gastrografin for abdominopelvis CT.

MATERIALS AND METHODS

A total of 98 patients referred to the Diagnostic

Imaging Department, Hospital Kuala Lumpur from

January to April 2009 for abdominopelvis CT with

Jostt vol 6.indd 5 7/22/10 10:08:25 PM

5


6

Journal of Science and Technology in the Tropics (2010) 6: 5-9

intravenous contrast enhancement were studied

using Siemens Somatom Sensation 16 scanner. The

study was approved by the Ethics Committee of the

Ministry of Health Malaysia.

Patients were divided into two groups of 49

patients each. The patients were randomly selected

by predefined inclusion and exclusion criteria to

participate in either of the two groups. The clinical

indications related to liver (30%), pancreas (30%),

renal (20%) and GIT (20%) for both groups did not

differ statistically with regard to their distribution (χ 2

test).

All patients were required to fast for at least 6

hours. Water or gastrografin (500 mL) was given at

60, 30 minutes and immediately before scanning. For

gastrografin 10 mL was diluted into 500 mL solution.

A scout image from xiphoid to the symphysis was

performed using a 16-detector row CT scanner

(Siemens, Somaton Sensation 16), followed by a

intravenous contrast bolus administration of Iopamiro

300 with a rate of 3 ml/s. Acquisition of scans was

obtained using the same protocol of 5-mm transverse

sections.

Two experienced radiologists independently

reviewed the images from each examination in

transverse and coronal planes on the 3 megapixel

workstation. The radiologists were blinded to the

clinical history of the patients. An incremental threepoint

scale (0 = worst, 0.5 = moderate, 1 = best) was

used to rate images from each examination for bowel

distension, homogeneity of the lumen, delineation

of the bowel wall, presence of artifacts and overall

image quality.

Overall bowel distension was graded from totally

collapsed (0) to maximal distension (1); homogeneity

of the lumen from massively inhomogeneous (0) to

completely homogeneous (1). The delineation of

the bowel wall was rated twice from indiscernible

(0) to clearly visible (1), initially against the

intraluminal contrast and then against the surrounding

extraintestinal tissue. The presence of artifacts was

rated as no artifacts (0) to serious image degradation

due to artifacts (1). Finally, overall image quality was

assessed from unreadable (0) to perfect (1).

To support the qualitative evaluation, additional

quantitative measurements were performed. The

maximum cross-sectional diameter of the antrum

of the stomach, horizontal part of the duodenum,

proximal jejunum, and terminal ileum were measured

perpendicular to the axis of the lumen using the

outer margins of the intestinal wall for each patient.

Attenuation in Hounsfield units (HU) of the lumen

and gastrointestinal wall was measured at the

same levels of the stomach, duodenum, jejunum,

and terminal ileum. Attenuation of the lumen was

measured by placing a region of interest (ROI)

within a well-distended segment of the small bowel

section. Attenuation of the wall was measured by first

zooming into the image section until the wall was

clearly visible, and then placing an ROI (minimum

diameter of 2 mm) over the bowel wall.

Mann-Whitney U-test was used to perform

statistical analysis of the differences in median scores

between water and gastrografin groups regarding

bowel distension, homogeneity of the lumen,

differentiation of the bowel wall against luminal

content and surrounding fat, the presence of artifacts

and overall image quality. Differences were considered

significant if p < 0.05; inter-observer agreement was

evaluated using weighted kappa statistics where a

kappa statistics > 0.75 was considered as excellent

agreement, 0.4-0.75 as fair to good agreement, and <

0.4 as poor agreement. Values near zero or less than

zero reflected only chance agreement.

For the quantitative analysis, the differences in

maximum diameters, HU values for the bowel lumen,

and contrast values between the neutral oral contrast

groups were compared using t-test; differences were

considered significant if p < 0.05

RESULTS

All patients tolerated the administration of oral

contrast medium. There was no vomiting, diarrhoea,

abdominal pain or allergic reactions to both the oral

contrast media. All patients drank the designated

amount of fluid within the given time. The images

were of diagnostic quality to be evaluated by two

independent radiologists with five years of experience.

Kappa analysis showed the inter-observer agreement

in the excellent category (k = 0.762).

The qualitative results were summarized in Figure

1. Delineation of the bowel wall from inside to outside

was significantly better using water compared to

gastrografin (p < 0.05). As for all the other criteria

there was no significant difference between water

and gastrogafin. The quantitative measurements are

summarized in Table 1. Diameters of the gaster and

Jostt vol 6.indd 6 7/22/10 10:08:26 PM



Location Oral contrast

Gastric

Duodenum

Jejunum

Iliem

Figure 1. Qualitative evaluation of abdominopelvis

CT images, water vs gastrografin

Table 1. Quantitative evaluation of gastrointestinal tract,

gastrografin vs water.

bowel were significantly better using water compared

to gastrografin (p < 0.05) while the other criteria

showed no significant difference using t-test and

Mann-Whitney U-test .

DISCUSSION

Diameter

(cm)

Attenuation

of bowel

wall (HU)

Attenuation

of bowel

lumen (HU)

Difference

wall to

lumen

Gastrografin 9.81 128.67 100.24 -28.43

Water 10.62 -7.46 52.56 60.02

p>0.05 p


8

JJournal of Science and Technology in the Tropics (2010) 6: 5-9

Figure 4. CT images of abdominopelvis with water

as the contrast medium.

reconstruction and computer workstations for

anatomical reconstruction have made it possible to

interpret every structure in abdominal CT studies,

including organs, vessels, bones and also bowel, in

a single examination (Fig. 4). Besides, ability of CT

that scans at a shorter time greatly eliminates artifacts

caused by peristaltic and respiratory movement. The

results were consistent with previous studies that

stated that positive oral contrast caused more streak

artifacts compared to water [11]. CT images can also

be reconstructed to get detail visualization of bowel

wall [18].

Subtle changes in the bowel wall and its

surrounding fat can be masked with the use of

positive oral contrast agent like gastrografin. The

reasons for this are enhanced bowel wall may have

the same attenuation as the positive contrast of

the lumen and high densities often occur due to

heterogeneous intraluminal distribution, leading to

artifacts with reduced visualization of the bowel wall

1. Foley W. D. and Kerimoglu U. (2004) Abdominal

MDCT; liver, pancreas, and biliary tract. Semin

Ultrasound CT MR 25: 122-144.

2. Horton K. M., and Fishman E. K. (2003) the current

status of multidetector row CT and three-dimensional

imaging of the small bowel. Radiol Clin North Am 41:

199-212.

3. Raptopoulos V., Davis M. A., Davidoff A., Karellas

A., Hays D., D’Orsi C. J. and Smith E. H. (1987)

Fat-density oral contrast agent for abdominal CT.

Radiology 164: 653-656.


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and surrounding fat [16]. This may be the reason

for increasing interest in the use of low attenuation

contrast for a wider range of indications. Water is the

primary choice; advantages include low cost, wide

availability, natural and safe for anyone without any

complications [7].

Water showed significantly better contrast between

bowel wall and lumen and for other criteria in

qualitative analysis, including homogeneity in lumen,

delineation of bowel wall and overall quality image,

consistent with other previous studies. Combination

of water that acts as negative oral contrast agent and

intravenous contrast agent that acts as positive contrast

agent give a better appreciation of mural detail.

Mucosal folds can be visualized clearly and actual

bowel wall thickness can be determined. However,

the limitation of using water as an oral contrast agent

is that the patient needs to consume a large quantity

of water, especially for those in-patients who are very

ill. Some of them even vomit after trying to take more

water. Previous study reported that there were cases

of water toxicity after consuming large amount of

water by patients who had chronic renal failure and

congestive heart failure [7]. Water is not a suitable

oral contrast for these types of patients.

In summary, this study showed that water was

comparable to gastrografin as an oral contrast

medium. Qualitative evaluation showed significantly

superior bowel distension and contrast between bowel

wall and lumen. Besides, quantitative measurements

showed better distension of gaster and bowel. Water

is therefore a better choice as oral contrast medium

since it is inexpensive and safe to consume.

4. Turetschek K., Schober E., Wunderbaldinger P.,

Bernhard C., Schima W., Puespoek, A., Vogelsang

H., Moeschl P. and Mostbeck G. (2002) Findings at

Helical CT-Enteroclysis in Symptomatic Patients

With Crohn Disease: Correlation With Endoscopic

and Surgical Findings. Journal of Computer Assisted

Tomography 26: 488-492.

5. Horton K. M., and Fishman E. K. (2004)

Multidetector-row computed tomography and

3-dimensional computed tomography imaging of

small bowel neoplasms: current concept in diagnosis.

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Journal of Computer Assisted Tomography 28: 106-

116.

6. Horton K. M., and Fishman E. K. (2001) Multidetector

row CT of mesenteric ischemia: can it be done?

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7. Winter T. C., Ager J. D., Nghiem H. V., Hill R. S.,

Harrison S. D. and Freeny P. C. (1996) Upper

gastrointestinal tract and abdomen: water as an orally

administered contrast agent for helical CT. Radiology

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8. Hori S., Tsuda K., Murayama S., Matsushita M.,

Yukawa K. and Kozuka T. (1992) CT of gastric

carcinoma: preliminary results with a new scanning

technique. Radiographics 12: 257-268.

9. Spilde J., Lee F. T., Chosy S. G., Krupinski E. A.,

Kuhlman J. E. and Yandow M. R. (1999) Evaluation

of an experimental low-attenuation gastrointestinal

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in an animal model. Acad Radiol 6: 94-101.

10. Thompson S. E., Raptopoulos V., Sheiman R. L.,

McNicholas M. M. J. and Prassopoulos P. (1999)

Abdominal helical CT: Milk as low-attenuation oral

contrast agent. Radiology 211: 870-875.

11. Matsuoka Y., Masumoto T., Koga H., Suzuki K.,

Ushimi T., Terada H., Tamura A., Yokoyama Y., Abe

K. and Kamata N. (2000) Positive and negative oral

contrast agents for combined abdominal and pelvic

helical CT: first iodinated agent and second water.

Radiation Medicine 18: 213-216.

12. Berther R., Patak M. A., Eckhardt B., Erturk S. M.

and Zollikofer C. L. (2008) Comparison of neutral

oral contrast versus positive oral contrast medium in

abdominal multidetector CT. Eur Radiol. 18: 1902-

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13. Balfe D. M., Koehler R. E., Karstaedt N., Stanley R.

J. and Sagel S. S (1981) Computed tomography of

gastric neoplasm. Radiology 140: 431-436.

14. Komaki S. and Toyoshima S. (1983) CT’s capability

in detecting advance gastric cancer. Gasrointest Radiol

8: 307-313.

15. Macari M. and Balthazar E. J (2001) CT of bowel wall

thickening: Significance and pitfalls of interpretation.

AJR 176: 1105-1116.

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status of multidetector row CT and three-dimensional

imaging of the small bowel. Radiol Clin North Am

41:199–212.

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MDCT of abdominal trauma. pp. 185-195. Springer,

Milan.

18. Gossios K. J., Tsianos E. V., Demou L. L., Tatsis C. K.,

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Jostt vol 6.indd 10 7/22/10 10:08:33 PM


Journal of Science and Technology in the Tropics (2010) 6: 11-16

Effect of colchicine on tissue culture derived plants of Zingiber officinale Rosc.

and Zingiber officinale var. rubrum Theilade

Lindayani 1 , K. Norzulaani 2 , H. Ibrahim 2 and Noorsaadah Abd Rahman 3

1 Department of Food Technology, Faculty of Agricultural Technology, Soegijapranata Catholic University,

Pawiyatan Luhur IV/1 Bendan Duwur, Semarang 50234, Indonesia

2 Institute of Biological Sciences, 3 Department of Chemistry, Faculty of Science, University of Malaya,

Lembah Pantai, 50603 Kuala Lumpur, Malaysia

(Email: lindayani@unika.ac.id)

Received 04-01-2010; accepted 01-02-2010

Abstract The aim of this study was to assess the effect of colchicine on the morphology and histology of

gingers in view of polyploidisation. Rhizomes of ginger were treated separately with three concentrations of

colchicine (0.5%, 1.0% and 2.0% w/v) for 30, 60 and 120 minutes. For morphology study, the fresh weight

of rhizome (FWRH), length of root (RLEN) and plant height (PH) were measured. Histological analysis was

done on shoot buds and rhizomes. All the parameters tested gave significant result. An increasing trend was

observed with colchicine concentration up to 1.0%, but decreased at 2.0%. Histological analysis showed that

the cell size of the shoot tip of Zingiber officinale Rosc. treated with colchicine (2.0%) for 120 minutes was 1.5

times larger than the control. For Zingiber officinale var. rubrum Theilade, the cell size was similar for treated

and control shoot tips. Over all this study showed that polyploidisation might occur with colchicine treatment

of various concentration and incubation time.

Keywords plant morphology – histology – polyploidisation – ginger

INTRODUCTION

Ginger, Zingiber officinale Rosc (Zingiberaceae), has

been used as a spice both in the East as well as in

the West since time immemorial [1]. In Peninsular

Malaysia there are at least three local races, namely

halia betul (true ginger), halia bara (red ginger) or

alternatively halia padi, and halia udang [2]. Both

halia bara and halia padi are distinguished from

halia betul from their small rhizomes. Halia udang is

probably extinct but halia bara differs slightly from

halia padi by its externally red rhizome. Halia bara

and halia padi are more pungent than the normal

ginger and are mainly used in traditional medicine

[2,3]. Pharmacological studies have shown that

ginger rhizomes are effective for the recovery of

intestinal disorder [4] and salivary secretion [5], for

stimulating the vasomotor and respiratory centres,

and for lowering serum and hepatic cholesterol levels

[6,7].

Apart from the normal mitotic processes, polyploidy

can be induced by treatment with colchicine [8-10].

The in vitro induction of polyploids with colchicine

has been reported in many plant species [11-15]. The

increase in the ploidy level could occur relatively

easily and may lead to an overall enlargement of plant

organs, for example cells, stomata, leaves, flowers,

fruits and seeds [8].

Tetraploid gingers (Z. officinale) strains “4x

Kintoki”, “4x Sanshu” and “4x Phillippine Cebu 1”

have been produced by soaking shoot tip explants

in a colchicine solution (0.2%, w/v). The induced

tetraploid gingers were much bigger in plant and

rhizome size than the diploids [14]. In addition,

the tetraploid ginger had higher pollen fertility and

germination rates than the diploids. However, seed

settings in the tetraploid ginger have not yet been

reported [16].

Colchicine prevents spindle formation at prophase,

precludes a nuclear mitosis, delays chromosomal

separation, inhibits daughter nuclei, and effectively

blocks the cleavage processes. Hence, when root tips

Jostt vol 6.indd 11 7/22/10 10:08:33 PM

11


12

Journal of Science and Technology in the Tropics (2010) 6: 11-16

or other growing plant parts were placed in appropriate

concentration of colchicine, the chromosomes of the

treated cells duplicated without spindle formation

and the cytoplasmic phase of cell division would not

occur [17].

In general, colchicine is used as an antimitotic

agent in plants. It influences the duplication of the

number of chromosome. The effect of colchicine

could be determined through the plant morphology.

The present study was carried out to examine the

effect of colchicine on the morphology and histology

on tissue culture derived ginger plants Zingiber

officinale and Zingiber officinale var. rubrum.

MATERIALS AND METHODS

Plant Material

The rhizomes of Z. officinale and Z. officinale var.

rubrum used in this study were obtained from the

local wet market. The specimens were authenticated

by Halijah Ibrahim from the Institute of Biological

Sciences, University of Malaya.

Application of colchicine

Colchicine solutions were cooled after autoclaving

and were freshly prepared before use. Fourteen to

Table 1. Combinations of treatment with colchicines

concentration (C) and incubation time (T) used for

in vitro derived shoot buds of ginger.

Concentration of

Colchicines (%)

CO (0.0)

C1 (0.5)

C2 (1.0)

C3 (2.0)

Incubation time

(minutes)

TO (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

T0 (0)

T1 (30)

T2 (60)

T3 (120)

Number of

Shoot bud

16

16

16

16

14

14

14

14

14

14

14

14

14

14

14

14

sixteen shoot bud pieces were used for each

treatment combination (Table 1). These buds were

soaked in sterile distilled water (control), 0.5%

(w/v), 1.0% (w/v) or 2.0% (w/v) aqueous colchicine

solution in a 250-mL Erlenmeyer flask placed on an

orbital shaker (90 rpm) at 24±2°C. The shoot buds

were incubated according to the selected time (Table

1). Following that, each bud was dried on sterile

filter paper and subsequently placed in MS semisolid

initiation media under a 16 hours photoperiod at

3500 lx using white fluorescent tubes. The

temperature was maintained at 24±2°C in the growth

room. After about five months (5 th subculture),

the surviving colchicine-treated plantlets were

transferred to sterile vermiculite placed in plastic

polybags (3″ x 6″) and kept in a growth room under

a 16:8-h light:dark photoperiod at 24±2°C for 4

months before transferring to the nursery.

Growth maintenance of colchicine treated plants

After the plantlets had rooted and reached 3-5 cm

in height with well expanded leaves, they were

transplanted into pots filled with soil mix consisting

of sand:peat:top soil (3:2:1), vermiculite and fertilizer

(N:P:K at the ratio of 15:15:15) after washing off the

agar with tap water. To avoid fungal infection, the soil

mix in the pot was drained with 1% (w/v) Thiram ®

over night before planting. For pest control, plants

were sprayed with 1.0 mL of Plusbon 250 ® diluted

with 5 L water. Plants were irrigated daily and 1.0 g

of foliar fertilizer (Agrospray 63 ® ) diluted in 5 L tap

water was sprayed monthly for supplement.

Measurement of plant morphology

The plants were monitored every month. They

were maintained in the nursery for one year before

harvesting and analysis. For each treatment, fresh

weight of rhizome (FWRH) and plant height (PH)

were measured. Length of the root (RLEN) was

measured randomly by using Image-Pro ® Express

Version 4.5 (Media Cybernetics).

Histological analysis

The shoot buds were fixed in FAA (formalin, acetic

acid glacial, ethyl alcohol: 5:5:90), then progressively

dehydrated in ethanol series, and finally infiltrated in

paraffin. Longitudinal sections (10-20 µm thick) of

paraffin-embedded materials were obtained using a

Jostt vol 6.indd 12 7/22/10 10:08:34 PM


otary microtome. The sections were stained with

a mixture of 1% (v/v) safranine and 1% (v/v) fast

green and mounted with Canada Balsam [18]. Size

of cells was measured using a micrometer eyepiece

where 0.01 mm equals to 5 mm (under x100 and x400

magnification). This work was done at the laboratory

of plant histology, Biological Faculty, University of

Gadjah Mada, Yogyakarta, Indonesia.

Statistical analysis

Analysis of variance (ANOVA), performed with the

program SAS (SAS ® proprietary software Release

6.02), of factorial design was used to test the effect of

the concentrations of colchicine and incubation times.

Duncan Multiple Range Test (DMRT) at p


14

Journal of Science and Technology in the Tropics (2010) 6: 11-16

Plant height measured for Z. officinale (V1) showed

the highest reading (48.24 cm) for treatment C1T1

compared to the other treatments. C1T1 treatment


Figure 2. Rhizome of Zingiber officinale, V1, C0T0

(1) and Zingiber officinale var. rubrum, V2, C0T0

(2) obtained from control; and rhizome of Zingiber

officinale, V1, C3T2 (3) and Zingiber officinale var.

rubrum, V2, C1T1 (4) after treatment. C0T0 = Control;

C3T2 = colchicine conc. 2%, incubation time 60 min;

C1T1 = Colchicine conc. 0.5%, incubation time 30 min.


for Z. officinale var. rubrum (V2) exhibited similar

result for the control (43.38 cm) and treated group

(43.16 cm) (Table 3.). Roots for Z. officinale (V1)

were shorter compared to Z. officinale var. rubrum

(V2). The best result for Z. officinale was produced

by treatment C1T1 while for Z. officinale var. rubrum

the longest root measurement was obtained when the

shoot bud was briefly dipped in treatment C1T0.

To date, there are no reports on the morphology of

ex vitro colchicine treated plants as described at this

study. Based on this study, an increasing trend for fresh

Table 3. Fresh weight of rhizome (FWRH, g), PH,

plant height (PH, cm) and root length (RLEN, cm) from

combination of variety, concentration of colchicine and

incubation time after transferring to the nursery. V, Variety

(V1, Zingiber officinale; V2, Zingiber officinale. var.

rubrum ); C, concentration of colchicine; T, incubation time.

Different alphabets show significant differences (p


weight of rhizome, plant height and root length was

observed with increasing colchicine concentration up

to 1.0% but decreased at 2.0%. For incubation time,

results were not consistent. Based on morphological

characteristics (fresh weight of rhizome, plant height

and root length) for treated plants, significant results

were found in all the data. When colchicines was

used, various organs of tetraploid gingers become

huge in appearance i.e. plant height, the size of leaves

and rhizomes, compared to diploids [14].

Histological analysis of shoot tips

Preliminary results showed that the cell size of the

shoot tip treated with colchicine (2.0%, C3) and

incubation time (T3, 120 minutes) was 1.5 times

larger than the control (C0) for sample V1 (Fig. 3).

For sample V2, the treated and control shoot tips

had similar cell size (Fig. 4).

From this preliminary histological analysis, the

effects of colchicine concentration were observed for

sample V1, but the result obtained did not confirm

or prove that polyploidisation was induced by the

treatments. Future work on quantitative analysis is

needed.

CONCLUSION

The effect of colchicine treatment on Z. officinale

and Z. officinale var. rubrum could be observed in

the morphology and histology of the treated plants.

An increase in the fresh weight of the rhizomes of

the treated plants was observed as compared to

the control. However, the results from qualitative

histological studies were inconclusive.


Figure 3. Micrographs of a section of the shoot tips of Zingiber officinale (V1). C0 (control) and C3 (treatment

with colchicine concentration 2.0% and incubation time 120 minutes).


Figure 4. Micrographs of a section of the shoot tips of Zingiber officinale var. rubrum (V2). C0 (control) and C1

(treatment with colchicine concentration 0.5% and incubation time 30 minutes).

Jostt vol 6.indd 15 7/22/10 10:08:40 PM

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Journal of Science and Technology in the Tropics (2010) 6: 11-16


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Journal of Science and Technology in the Tropics (2010) 6: 11-16

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Journal of Science and Technology in the Tropics (2010) 6: 17-21

Diversity and abundance of Dacinae fruit flies (Insecta: Diptera: Tephritidae) in

Chini 2, Runchang and Sungai Bebar, Pahang, Peninsular Malaysia

Hoi Sen Yong1 , Y. F. Ng2 and Phaik Eem Lim1,3 1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

(E-mail: yong@um.edu.my)

2Centre for Insect Systematics, School of Environmental and Natural Resource Sciences,

Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 4300 Bangi,

Selangor D.E., Malaysia

3Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

ABSTRACT The diversity and abundance of Dacinae fruit flies of the family Tephritidae were studied at

seven localities in the Pekan District, Pahang Darul Makmur, in the east coast of Peninsular Malaysia. Male

fruit flies were collected in the morning by using the attractants methyl eugenol and cue-lure. Three species

(Bactrocera carambolae, Bactrocera papayae and Bactrocera umbrosa) of the methyl eugenol group were

recorded. Bactrocera carambolae was present in all seven localities, being most abundant in a forest fringe

locality near a river but uncommon in the two Sungai Bebar localities. Bactrocera papayae was present in four

localities with human habitation but absent in the three forest areas. It was most common in the two localities

in Chini 2 with village setting. Bactrocera umbrosa was found in three localities with typical village setting with

fruit trees of Artocarpus species. The cue-lure group was present in five of the seven localities studied. Four

species were present – Bactrocera infesta, Bactrocera melastomatos, Bactrocera nigrotibialis and Bactrocera

cf tau. Bactrocera nigrotibialis was the predominant species while B. infesta occurred in quite large number

in a river bank locality. The other two species (B. melastomatos and B. cf tau) appeared to be uncommon.

Bactrocera infesta represented the first documented record for Peninsular Malaysia.

Keywords Bactrocera species – Bactrocera infesta – methyl eugenol – cue-lure – Pekan District Pahang –

new record

INTRODUCTION

Fruit flies of the family Tephritidae are plant feeders.

Many members of the subfamily Dacinae are of great

economic and agricultural importance because of

damage caused to commercial fruits and vegetables.

The damage, if uncontrolled, may result in a total loss

of the crop. The subfamily is represented by some 800

species worldwide, with some 300 species in Asia/

South-east Asia [1, 2].

Dacinae fruit flies of the genus Bactrocera were

previously referred to the genus Dacus [1]. They may

be polyphagous or exhibit great specificity for host

fruits [3]. The degree of host specificity appears to

be related to the extent of genetic diversity [4]. These

fruit flies also show variability in their attraction to

male chemical lures [3].

Most of the studies on Dacinae fruit flies concern

the species of agricultural importance. The present

paper reports the diversity and abundance of Dacinae

fruit flies, as determined by application of male

chemical lures, in seven localities – urban areas in

Chini 2, and settlement and forest areas in Runchang

and Sungai Bebar – within the Pekan District of

the State of Pahang Darul Makmur, Peninsular

Malaysia.

MATERIALS AND METHODS

During a field trip in September 2009, we studied

the diversity and abundance of Dacinae fruit flies

in seven localities at Chini 2, Runchang and Sungai

Bebar. These localities are situated in the Pekan

District, Pahang Darul Makmur, in the east coast of

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Journal of Science and Technology in the Tropics (2010) 6: 17-21

Peninsular Malaysia. Chini 2 is a small town of the

Chini Felda scheme while Runchang is an Orang Asli

settlement in the Sungai Bebar area. The surrounding

of Sungai Bebar is peat swamp forest.

Male fruit flies were collected in the morning by

means of the sex attractants methyl eugenol (4-allyl-1,

2-dimethoxybenzene) and cue-lure (4-[4-(acetyloxy)

phenyl]-2-butanone). A small amount of each lure

was applied separately and away from each other on

the upper surface of a green leaf – the lures are very

effective and not affected by the leaf. Insects attracted

to the lures over a period of about 30 minutes were

collected with the aid of specimen tubes and plastic

bags. The specimens were preserved in ethanol in

the field and subsequently identified using current

literature and personal experience [1, 2, 5, 6].

Representative individuals were also photographed

in the field.

The diversity of the fruit flies was analysed using

BioDiversity Professional Ver 2.0 1997. Shannon-

Weiner index and Jaccard cluster analysis were

applied for this study.

RESULTS

The diversity and relative abundance of Dacinae

fruit flies collected in Chini 2, Runchang and Sungai

Bebar are listed in Table 1. Three species of methyl

eugenol group were present – Bactrocera carambolae

Drew and Hancock, Bactrocera papayae Drew and

Hancock and Bactrocera umbrosa (Fabricius). The

cue-lure attractant yielded four species – Bactrocera

infesta (Enderlein), Bactrocera melastomatos Drew

and Hancock, Bactrocera nigrotibialis (Perkins) and

Bactrocera cf tau.

For the methyl eugenol group all three species were

present in three localities, C2, R1 and R2. Two species

(B. carambolae and B. papayae) were collected in

C1 and only B. carambolae was found in R3, SB1

and SB2. Fruit flies of the cue-lure group were found

in five of the seven localities studied, not present in

C2 and R2 (Table 1). Bactrocera nigrotibialis was

the predominant species while B. infesta occurred in

quite large number in a river bank locality. The other

two species (B. melastomatos and B. cf tau) appeared

to be uncommon.

The Shannon-Weiner diversity index of the

Dacinae fruit flies revealed that R1 had the highest

H’ (0.58) followed by R2 (H’= 0.432) and C2 (H’=

0.401) (Table 1). Locations SB1 and SB2 yielded the

lowest H’ of 0.126 and 0.110 respectively (Table 1).

The Jaccard cluster analysis showed that R2 and C2,

and SB2 and R3 had respectively similarity of 100%

(Fig. 1).

DISCUSSION

An extensive trapping effort, using Steiner traps

baited with methyl eugenol or cue-lure and provided

with malathion, was conducted over 12-36 months in

the 1990s in Peninsular Malaysia and Thailand [7].

The trapped fruit flies were collected on a 1-2 week

Table 1. Species and number of Dacinae fruit flies collected in September 2009 by methyl eugenol

and cue-lure attractants from Chini 2, Runchang and Sg. Bebar (Pekan District, Pahang, Peninsular

Malaysia). C1, edge of secondary vegetation and fence of apartments in Chini 2; C2, abandoned village

house along main road and near housing estate in Chini 2; R1, near school and Orang Asli house with

orchard and secondary vegetation in Runchang; R2, near Orang Asli house in Runchang; R3, secondary

forest near river in Runchang; SB1, river bank near abandoned aquaculture area along Sg. Bebar; SB2,

forested area upstream from SB1 of Sg. Bebar.

Lure/species C1 C2 R1 R2 R3 SB1 SB2

Methyl eugenol

B. carambolae

B. papayae

B. umbrosa

Cue-lure

B. infesta

B. melastomatos

B. nigrotibialis

B. cf tau

22

58

2

28

26

5

12

16

8

1

1

1

4

6

12

63 1 3

Number of species 3 3 6 3 2 3 2

Shannon H’ 0.299 0.401 0.580 0.432 0.298 0.126 0.110

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1

40


asis. There were 12 stations in the state of Pahang,

Peninsular Malaysia – the exact localities were however

not stated, but presumably in agricultural areas. In

that exercise, only three species (B. carambolae,

B. papayae and B. umbrosa) of the methyl eugenol

group and a single species (B. cucurbitae) of the cuelure

group were recorded in Pahang.

In another study carried out in the Endau-Rompin

rain forest in southern Peninsular Malaysia from

August to November 1985, tephritid fruit flies were

collected by ‘Malaise trap’, hand collection, and

methyl eugenol baited and cue-lure baited traps

[8]. The lure-baited traps were set for four days per

month, with one trap for methyl eugenol and four for

cue-lure. This study yielded four Bactrocera species

and 28 non-Dacinae species – quantitative data were

not provided. The methyl eugenol baited traps yielded

fruit flies of the B. dorsalis complex – B. dorsalis

(Hendel) does not occur in Peninsular Malaysia and

the complex consists of many species [5]. The cuelure

baited traps produced two species, B. nigrotibialis

and B. tau. Bactrocera latifrons (Hendel), which is

not attracted to both methyl eugenol and cue-lure,

was collected in the Malaise trap, in addition to B.

dorsalis complex and B. tau.

The diversity of Bactrocera species in the present

study is comparable to studies using lure-baited traps

(with male attractants methyl eugenol and cue-lure). It

is also similar to studies employing the same method of

applying the lures on green leaves [9, 10]. The study in

Negeri Sembilan yielded two species (B. carambolae

and B. papayae) of the methyl eugenol group and

three species (B. melastomatos, B. nigrotibialis and

Jaccard Cluster Analysis (Simple Average Link)

0. % Similarity

50.

SB1

SB2

R3

R1

R2

C2

C1

100

B. tau) of the cue-lure group [9]. In Kelantan, the

two localities studied (Pantai Melawi and Selising)

supported different species and different density of

the common species – B. carambolae, B. papayae,

B. melastomatos in both localities, B. caudata only

in Pantai Melawi, and B. umbrosa only in Selising

[10]. The presence or absence of Bactrocera species

and their abundance are dependent on the availability

of their host plants/fruits [11]. For example, in the

present study B. umbrosa was found only in localities

with typical village setting. This species is rather

host specific [12], infesting fruits of Artocarpus spp.

which were not evident in the localities where it was

not collected.

In the extensive study carried out in Pahang [7],

the Bactrocera species showed seasonal abundance

in number. Bactrocera carambolae, B. papayae and

B. umbrosa had higher number in September than

other months, while B. cucurbitae showed a peak in

November. The present study carried out in September

2009 did not yield B. cucurbitae; it may be attributed

to the absence of host plants Cucurbitaceae which

were not evident in all the seven localities studied.

Of the cue-lure group of fruit flies, B. nigrotibialis

(Fig. 2) was the predominant species. It occurred in

large number in two forest areas but only a single

individual was encountered in an inhabited area. It

was present in the Endau-Rompin rain forest [8] but

not recorded in the extensive trapping carried out in

12 stations in Pahang [7]. This species is not confined

to the forest as it is present in the University of Malaya

campus (unpublished data).

An unexpected find in the present study was the

large number of B. infesta (Fig. 3) attracted to cuelure

in locality SB1 (river bank near an abandoned

Figure 1. Dendrogram of Dacinae fruit flies species

similarity of different localities in Chini District,

Pekan, Pahang . Figure 2. Male Bactrocera nigrotibialis.

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Journal of Science and Technology in the Tropics (2010) 6: 17-21

aquaculture area along Sg. Bebar). As far as we

are aware there is no published record of this

species for Peninsular Malaysia – we had however

encountered it before but not published. It was

originally described as Polistomimetus infestus

Enderlein 1920 and later treated as Dacus

(Pacifodacus) infestus [7]. It looks remarkably like

Callantra sphaeroidalis (Bezzi) but the first two

segments of the antennae are short [6]. The typelocality

is Sumatra; it has been recorded in Indonesia,

Thailand and Laos. It is a rufous species with

only a short median yellow vitta, a petiolate abdomen,

a very large apical spot in the wing, and a complete

black band along lower surface of face [6]. As far

as known, there is no published information on its

attraction to lure and host plants.

The present method of using chemical attractants

does not uncover the presence of tephritid species

that do not respond to such lures [3], e.g. B. arecae

and B. latifrons. In the study using four collection

methods in the Endau-Rompin rain forest, Malaise

traps accounted for 20 out of 32 species and hand

collection 12 species, compared to three species by

methyl eugenol and cue-lure [8].

In sum, different localities in the present study

yielded different composition and abundance of

Bactrocera species, due most likely to the presence

and availability of host plants/fruits. Of the seven

species present, B. infesta constitutes a new record

for Malaysia. The higher number of Bactrocera

species and Shannon H’ in localities R1, R2 and C2

are attributed to the presence and availability of host/

fruit and orchards in or near the localities. These

areas also showed higher similarity percentage as

indicated by Jaccard cluster analysis.

Acknowledgement – The field work was supported by

the research grant UKM-GUP-ASPL-07-04-048.

We thank our institutions (University of Malaya and

Universiti Kebangsaan Malaysia) for facilities and

financial support; and the Forestry Department of

Pahang Darul Makmur for permission to carry out field

research.

Figure 3. Male Bactrocera infesta.

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1. Drew R.A.I. (1989) The taxonomy and distribution

of tropical and subtropical Dacinae (Diptera:

Tephritidae). In Robinson A.S. and Hooper G. (Eds.)

Fruit Flies,Their Biology, Natural Enemies and

Control Vol. 3A pp. 9-14. Elsevier, Amsterdam.

2. Yong H.S. (1995) Genetic differentiation and

relationships in five taxa of the Bactrocera dorsalis

complex (Insecta: Diptera: Tephritidae). Bulletin of

Entomological Research 85: 431-435.

3. Yong H.S. (1996) Host specificity and response to

chemicals in Dacinae fruit flies (Insecta: Diptera:

Tephritidae). In Turner I.M., Diong C.H., Lim S.S.L.

and Ng P.K.L. (Eds.) Biodiversity and the Dynamics of

Ecosystems. DIWPA Series Volume 1:191-194.

4. Yong H.S. (1992). Host specificity and genetic

variability in Malaysian fruit flies (Insecta: Diptera:

Tephritidae). Proceedings of the National IRPA

(Intensification of Research in Priority Areas Seminar

(Agriculture Sector), Volume 1: Crops and Plants,

Kuala Lumpur, Malaysia, pp. 233-234.

5. Drew R.A.I. and Hancock D.L. (1994) The Bactrocera

dorsalis complex of fruit flies (Diptera: Tephritidae:

Dacinae) in Asia. Bulletin of Entomological Research

Supplement No. 2: 1-68.

6. Hardy D.E. (1973) The fruit flies (Tephritidae-Diptera)

of Thailand and bordering countries. Pacific Insects

Monograph 31: 1-353.

7. Clarke A.R., Allwood A., Chinajariyawong A., Drew

R.A.I., Hengsawad C., Jirasurat M., Krong C.K.,

Kritsaneepaiboon S. and Vijaysegaran S. (2001)

Seasonal abundance and host use patterns of seven

REFERENCES

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Bactrocera Macquart species (Diptera: Tephritidae)

in Thailand and Peninsular Malaysia. The Raffles

Bulletin of Zoology 49(2): 207-220.

Tan C.L., Md. Hanifah Y. and Chan H.F. (1994)

Diversity of fruit flies species (Diptera: Tephritidae)

collected in Endau-Rompin, Malaysia. In Yong H.S.

and Khoo S.G. (Eds.) Current research on tropical fruit

flies and their management pp. 27-31. The Working

Group of Malaysian Fruit Flies, Kuala Lumpur.

Yong H.S., Sofian-Azirun M. and Hashim R. (2009)

Dacinae Fruit Flies (Insecta: Diptera: Tephritidae) of

Lata Kijang Kenaboi, Negeri Sembilan, Peninsular

Malaysia. Malaysian Journal of Science 28 (4): 61-

64.

Yong H.S., Hashim R., Sofian-Azirun M. and Mat

Diah S.Z. (2009) Diversity and abundance of Dacinae

fruit flies (Insecta: Diptera: Tephritidae) in Pantai

Melawi and Selising, Kelantan, Peninsular Malaysia.

Malaysian Journal of Science 28 (Special Issue): in

press.

Yong H.S. (1994) Host fruit preferences in two

sympatric taxa of the Bactrocera dorsalis complex

(Insecta: Diptera: Tephritidae). In Yong H.S. and Khoo

S.G. (Eds.) Current research on tropical fruit flies and

their management pp. 1-8. The Working Group on

Malaysian Fruit Flies, Kuala Lumpur.

Yong H.S. (1988) Allozyme variation in the Artocarpus

fruit fly Dacus umbrosus (Insecta: Tephritidae) from

Peninsular Malaysia. Comparative Biochemistry and

Physiology 91B: 85-89.

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Journal of Science and Technology in the Tropics (2010) 6: 23-30

The mammal fauna of Pulau Singa Besar, Langkawi, Kedah,

Peninsular Malaysia

Lim Boo Liat1 , Norhayati Ahmad2,3 , O. Noor Alif Wira4 , Shahrul Anuar Md. Sah5 1Jalan Koop Cuepacs 3 E, Taman Cuepacs, 43200 Cheras, Selangor, Malaysia

2School of Environment and Natural Resource Sciences, Faculty of Science and Technology,

University Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

3Institute for Environment & Development (LESTARI) , Universiti Kebangsaan Malaysia,

43600 UKM Bangi, Selangor, Malaysia

4Department of Wildlife and National Parks, Penang, Malaysia

5Department of Biological Science, Universiti Sains Malaysia, Penang, Malaysia

(Email: chizlim54@yahoo.com)

Abstract Studies on the mammal fauna of Pulau Singa Besar, Langkawi were carried out during 2001

and 2002. A total of 51 species – 21 non volant and 30 volant species – from past and recent studies were

recorded. Two species, the Smooth otter (Lutrogale perspicillata) and Indian False vampire (Megaderma

lyra) are new records for the Langkawi Archipelago. The species diversity between the non volant and volant

mammals is discussed.

Keywords mammals – Malaysia – bats – diversity – island

INTRODUCTION

Pulau Singar Besar, Langkawi is one of the 104 islands

in the Langkawi Island archipelago. It is a permanent

forest reserve, uninhabited, and was established as a

bird and animal sanctuary in 1990. The island consists

of various pristine forest types, such as dipterocarp

lowland tropical forest, mangrove coastal forest, and

sandy beach forest. In terms of the mammal fauna,

the island has been known for the high density of

mousedeer (Tragulus kanchil, T. napu), wildboar

(Sus scrofa), and Long-tailed macaque (Macaca

fascicularis).

Not much was known of its mammal species

diversity until the late 1980s. A survey was carried out

on the vertebrate fauna and 17 species of mammals

were recorded [1]. After a lapse of 10 years, more

comprehensive studies on the vertebrate fauna

(amphibian and reptile, mammal, bird) were carried

out at different periods of 8–10 days each in 2001

and 2002 under the purview of the Department of

Wildlife and National Parks (DWNP). We report here

the results of the 47 mammal species.

MATERIALS AND METHODS

Pulau Singa Besar is located south of the main

Langkawi island, wedged in between the small island

of Pulau Beras Basah on the west and the larger

island of Pulau Dayang Bunting on the east (Fig.

1). The island is 11.3 km 2 in area, reaches 270 m in

elevation and is nearly entirely covered by pristine

dipterocarp forest. The island is traversed by steep

valleys drained by seasonal streams. Limestone

outcrops are prominent on the north-eastern part of

the island. Caves of varying depths and widths occur

in this limestone area. There are three clear water

streams that flow to the east of the island, Sg. Boton

and Sg. Pantai, while Sg. Sepai flows to the northern

coast.

The island, as in the Kadawi region, experiences

dry periods between December-April. During the

north-east monsoon, the island is exposed to torrential

rain and storms. Day temperature varies from 27°C

under shade and 37ºC in the open on the beach. Night

temperature is around 24–27°C. It is hot and humid

most of the time [1].

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Journal of Science and Technology in the Tropics (2010) 6: 23-30

Figure 1. Map of Pulau Singa Besar and the trails where trapping of mammals was carried out.

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The field team comprised eight members, six of

whom were from the DWNP, Kuala Lumpur. Three

field trips were carried out: 2-14 May 2001, 2-8

February 2002, and 2-13 June 2002. Trapping of

the mammals was carried out at designated trails:

Mangrove boardwalk (MBW), Cicada trail (CT),

Perdana trail (PT), Dusun trail (DT), Sungai Sepai

trail (SS) and the Staff quarters (SQ). Trappings were

carried out by four field workers along two trails for

each of the field trip. Fifty collapsible traps and two

harp traps were deployed at each sampling site. Thus,

the three field trips covered all the six trapping sites. All

mammals captured were processed, i.e. measurements

were taken and reproductive conditions noted. While

visiting the various traps during the day and night, any

nocturnal and diurnal animals sighted were noted and

identified up to the species level whenever possible.

Voucher specimens for some of the volant mammals

(1–4 individuals) were collected and preserved using

ethanol and some of the non-volant species were

skinned for voucher specimens. All the voucher

specimens were deposited in the museum collection at

the Institute of Biodiversity, Bukit Rengit, Lanchang,

Pahang, Peninsular Malaysia.

Mangrove boardwalk (MB)

The mangrove boardwalk meanders through a patch

of mangrove forest next to the Staff quarters (SQ)

and Interpretation Center (IC) building just before

entering the forest. Rat traps were laid throughout the

200-m length of the boardwalk. Harp traps were set at

2 m apart at the mid-point on the boardwalk.

Cicada trail (CT)

This trail starts from a small section of the boardwalk

proceeding along the sandy beach. It is about 2 km

long. The trail is flanked on the left by disturbed

forest and the right by the sea coast. It is a popular trail

among tourists during weekends and public holidays.

Traps were laid in the forest and at the fringe. Harp

traps were set in the forest.

Perdana trail (PT)

The trail starts from the end of CT, heads south-west

through various forest types (scrub, secondary and

primary forests), meanders to northeast and ends at

Sg. Sepai Valley (SS). The terrain is undulating with

some parts very steep. The trail is about 6 km from

PT to SSV. As the trail is very long, the 50 traps were

divided into two areas along the trail at 25 m interval.

The first 25 traps were spread out along 1.5 km from

the beginning of the CT and ended at the base of the

steep point at about 800 m high; the second set of 25

traps were laid just below the steep base and ended at

the SSV. The harp traps were set at the midpoint of PT

and the other at the midpoint of SSV.

Dusun trail (DT)

The trail starts at 1.5 km of the PT, takes a 45 degree

turn to the right and descends another 0.5 km to a

small orchard of mostly durian trees. Another way of

getting to the DT is via boat during high tide through

the mangrove forest. Traps were laid in the plantations,

fringe of mangrove and along forest trails and stream

banks. Harp traps were set in the plantations and also

in the entrance of crevices of rock boulders in the

area.

Sungai Sepai valley (SS)

This area is one of the campsites for visitors to the

island. The forest here is disturbed primary forest

edged by a small stretch of mangrove forest. Deeper

into the secondary forest is pristine forest habitat. A

large forest stream, about 1-2 m wide undulating to

a stretch of limestone rocks, provides the source of

clean water for the campsite. Traps and harp traps

were set in the primary part of the forest.

Staff quarters (SQ)

There are four buildings making up the staff quarters

next to the MW and IC near the jetty. Traps and harp

traps were set at a stretch of disturbed forest behind

the SQ.

RESULTS AND DISCUSSION

A total of 47 mammals was recorded from the six

study sites from the three field trips in 2001 and 2002

on the island. This comprised 18 species of nonvolant

mammals from 12 families and 29 species of

volant mammals from eight families (Appendix 1).

The greater number of volant species was probably

due to the availability of roosting sites (crevices of

rock boulders, caves, hollow trees) in the island. The

low species diversity of the non volant mammals in

island ecosystem is generally low and Pulau Singa

Besar is no exception. This is also shown on studies

of other islands, for example, 45 mammal species

(including 19 bat species) in Pulau Tioman, Pahang

and 23 species (including 15 bat species ) in Pulau

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Journal of Science and Technology in the Tropics (2010) 6: 23-30

Perhentian [2-4]. This is due to the isolation from

the mainland where emigration and immigration are

marginal and also limited sizes of the landmass. In

the case of arboreal species (bats and birds), they

are very mobile and if it is within their flight range,

the island would serve as part of their transit shelters

for resident and migrant animals, as well as for food

resources.

Among the six study sites, higher species richness,

ranging from 21–38 species were recorded at CT,

PT, DT, and SSV. The higher number of species is

associated with the diversity of habitats (scrub,

secondary and primary forests) found in each of these

study sites. The markedly lower species richness

recorded at MBW (8 species) and SQ (12 species)

was probably due to the less diverse habitat of the

coastal mangrove forest as compared to the other

sites (Appendix 1).

Non-volant mammals

All the 18 mammal species recorded in the island

(Appendix 1: No. 1-18) are common species, except

the Smooth otter (Lutrogale perspicillata), which is a

new record for Langkawi. Most abundant among the

18 species were the tree-shrew, long-tailed macaque,

plantain squirrel, three species of field and forest rats,

wild boar and mouse-deer (Appendix 1: Nos. 4, 5,

7, 9-11, 17, and 18). The least encountered was the

White-toothed ground shrew (Crocidura fuliginosa)

(Appendix 1: No 1). This species is a tiny, burrowing

animal. Pit fall traps have been proven to be the most

suitable trapping technique to obtain this species [5].

Previous study [1] recorded 15 species of nonvolant

mammals on Pulau Singa Besar, of which, four

species were additional records for the island: the Slow

loris (Nycticebus coucang), Black giant tree squirrel

(Ratufa bicolor), Eurasian otter (Lutra lutra) and the

Three-striped palm civet (Arctogalidia trivirgata).

With these, the non-volant mammals increased to 22

species on Pulau Singa Besar. Of these, 20 species

were recorded on the main Langkawi island [6],

except Niviventer cremoriventer and Lutrogale

perspicillata. However, all the 22 species recorded

from Pulau Singa Besar are common in the mainland

of Peninsular Malaysia, except the Eurasian otter.

Volant mammals

The 29 species of bats recorded on the island comprised

seven species of fruit bats (Pteropodidae) and 22

species of insectivorous bats from seven families:

Emballonuridae, Nycteridae, Megadermatidae,

Rhinolophidae, Hipposiderosidae, Vespertilionidae

and Molossidae (Appendix 1: No. 19–47).

Fruit bats

All the seven species of fruit bats are common on the

island; the Lesser Dog-faced fruit bat (Cynopterus

brachyotis) and the Cave fruit bat (Cynopterus

Eonycteris spelaea) (Appendix 1; No. 20, 22) were

more common than the rest (Appendix 1: No. 19,

21, 23-25). Both these species were netted in all the

six study sites with greater frequency of captures

(27, 17 individuals respectively) compared to the

others which were netted between 2-4 study sites and

with less frequency of captures, ranging from 4–12

individuals.

Mustaffa Babjee [1] recorded four species of fruit

bats on the island. One of them, the Island Flying fox

(Pteropus hypomelanus) was a new locality record

for Langkawi, which increased the fruit bat fauna to

eight species from the island to date, compared to five

species in the main Langkawi island [6]. However,

all the eight species recorded on the island are also

common in lowland and hill forests in mainland

Peninsula Malaysia.

Insectivorous bats

The species richness of this group was reflected by the

troglodytic habits where the roosting environments

are available on the island. At least 12 species

(Appendix 1: No. 29-33, 36-40,42, 45) were cave

dwellers [7-9]. Among the 22 species, three species,

the Intermdiate Horseshose bat (Rhinolophus affinis),

Bicolored Roundleaf bat (Hipposideros bicolor) and

Common Roundleaf bat (Hipposideros galeritus),

were common with high frequency of captures (44,

38, and 27, respectively). Of particular interest was a

species with a single specimen netted, the Indian False

vampire (Megaderma lyra), which is uncommon.

This species is carnivorous in habit [6].

Fourteen of the 22 species on the island were also

recorded on the main Langkawi island [6]. On the

mainland of Peninsular Malaysia, all the 22 species

are common in lowland and hill forests.

Reproductive status

The examination of pregnant females was based

on visible observation of advance pregnancy of

the animals. This method was used to conserve the

species from being sacrificed for detail examination

Jostt vol 6.indd 26 7/22/10 10:09:03 PM


of the reproductive tracts, and also to prevent the

animal from being overly stressed by holding it too

long before release.

A total of 35 species, comprising eight species of

non-volant (63 individuals) and 27 species of volant

mammals (138 individuals), was examined in May

2001 and February and June 2002 (Table 1). Of the

non-volant mammals, two species (9 individuals)

were not gravid. For the females of the other six

species (Table 1: No. 1-2, 4-6, 8) with 54 individuals,

about 33% (18 individuals) were visibly pregnant.

The number of individuals pregnant during the three

periods showed no marked difference, being 5, 7, and

6 respectively.

Of the 27 species of volant mammals examined,

13 species (26 individuals) were not gravid. In the

other 14 species (Table 1: No. 9-10, 12-14, 19, 21,

24-25, 28, 30, 33-35) with 112 individuals, 36.7%

(41 individuals) were visibly pregnant. The number

of individuals pregnant during May 2001, February

and June 2002 was 15, 4, and 22 which suggests that

there was a high birth trend in May and June.

Table 1: Number of visibly pregnant females of non-volnt and volant mammals of Pulau Singa Besar, Langkawi,

Kedah, Peninsular Malaysia.

No. Species

No. Indiv.

Examined

2-14 May

2001

2-12 Feb

2002

2-13 Jun

2002

NON-VOLANT MAMMALS

1 Tupaia glis 7 2 2

2 Callosciurus notatus 7 1 1

3 C. caniceps 5

4 Rattus tiomanicus viclana 21 4 3 5 12

5 Rattus exulans 7 1 1

6 Leopoldamys sabanus 8 1 1

7 Niviventer crimoriventer 4

8 Maxomys surifer 4 1 1

VOLANT MAMMALS

9 Rousettus amplexicaudatus 2 1 1

10 Cynopterus brachyotis 12 1 3 4

11 C. horsfieldii 2

12 Eonycteris speleae 9 1 2 3

13 Penthetor lucasi 8 1 1

14 Macroglossus minimus 5 1 1

15 Emballonura monticola 4

16 Taphozous melanopogon 1

17 Nycteris tragata 4

18 Megaderma spasma 1

19 Rhinolophus affinis 24 4 2 5 11

20 R. trifoliatus 3

21 R. coelophyllus 3 1 1

22 R. pusilllus 2

23 R. lepidus 3

24 Hipposideros bicolor 21 4 1 6 11

25 H. galeritus 11 1 2 3

26 H. cinareus 1

27 H. diadema 2

28 H. larvatus 5 1 1

29 Myotis muricola 1

30 M. hasseltii 1 1 1

31 Murina suillus 1

32 Kerivoula papillosa 1

33 K. intermedia 4 1 1

34 Scotophilus kuhli 4 1 1

35 Tadarida mops 3 1 1

Total 201 20 11 28 59

Jostt vol 6.indd 27 7/22/10 10:09:04 PM

Total

27

Journal of Science and Technology in the Tropics (2010) 6: 23-30


28

Journal of Science and Technology in the Tropics (2010) 6: 23-30

Conclusion

The high species richness of the chiropteran

fauna in Pulau Singa Besar could be due to: (1)

the island with its pristine forest and surrounded

by limestone outcrops and traversed by valleys

with seasonal streams, provides abundant

roosting sites, such as caves, crevices of rock

boulders, tree holes, etc.; and (2) bats could fly

from neighbouring Pulau Basah on the west and

the larger Pulau Dayang Bunting on the east.

The non volant mammals are mostly terrestrial

residential species where emigration-immigration

is limited. Thus, the low species richness of this

1. Mustaffa Babjee A. (1990) Pulau Singa Langkawi:

bird and animal sanctuary. Department of Veterinary

Sciences, Ministry of Agriculture, Malaysia.

2.

3.

4.

5.

Lim B.L., Kelvin K.P.L. and Yong H.S. (1999) The

terrestrial mammals of Pulau Tioman, Peninsular

Malaysia, with a catalogue of specimens at the Raffles

Museum, National University of Singapore. Raffles

Bulletin of Zoology 6: 101-123.

Yong H.S. (1974) Diversity and abundance of small

mammals in two Malaysian offshore islands. Malayan

Nature Journal 27: 27-29.

Tamblyn A., Malley R., Turner C. and Hughes T.

(2009) The bat fauna (Mammalia, Chiroptera) of

Pulau Perhentian, Peninsular Malaysia. Malayan

Nature Journal 61(1): 10-22.

Nor Zalipah M., Juliana S. and Lim B.L. (2007) A note

REFERENCES

group of animals in the island ecosystem is a

natural phenomenon [10].

With the additional four species, Pteropus

hypomelanus, Nycticebus coucang, Lutra lutra

and Ratufa bicolor recorded in earlier study [1],

the mammal fauna of the island now stands at 51

species.

Acknowledgements – We are grateful to the staff of

DWNP, particularly the Director-General, Dato’ Musa

Nordin (retired) for his support; and the General Manager

of the Langkawi Development Authority (LADA), for

supporting the logistic in the field.

on the arboreal monkey-footed rat in a pit-fall trap.

Journal of Wildlife and Parks 24: 63-65.

6. Medway Lord (1983) The wild mammals of Malaya

(Peninsular Malaysia) and Singapore. 3rd Edn. Oxford

University Press, Kuala Lumpur.

7. Medway, Lord (1965) Identification of Malaysian

cave bats. Malayan Nature Journal 19: 88-107.

8. Lim B.L. (1967) Abundance and distribution of

Malaysian bats in different ecological habitats. Federal

Museum Journal (N.S.) II (1966): 61-76.

9. Norhayati A., Shukor M.N., Shahrul Anuar M.S.,

Nurul Ain E. and Norzalipah M. (2007) Note on the

bats of Gua Kelawar, Langkawi Island, Kedah. Journal

of Wildlife and Parks 24: 1-6.

10. Alle W.C. and Schmidt X. (1951) Ecological animal

geography. Chapman and Hall Ltd., London.

Jostt vol 6.indd 28 7/22/10 10:09:05 PM


Appendix 1: Mammal species recorded on Pulau Singa Besar, Langkawi, Kedah, Peninsular Malaysia. (MB=Mangrove

Boardwalk, CT=Cicada Trail, PT=Perdana Trail, DT=Dusun Trail, SS=Sungai Sepai Valley, SQ=Staff Quarters;

S=Sighted, S-TR=Sighted and track signs, R=Released).

No. Order/Family/Species

INSECTIVORA

Soricidae

MB CT PT DT SS SQ

Total

Male Female

Voucher

No.

1 Crocidura fuliginosa 1 1 0 35

DERMOPTERA

Galeopterida

2 Galeopterus variegatus S - 1 1 -

PHOLIDOTA

Manidae

3 Manis javanicus S -1 1 -

SCANDENTIA

Tupaiidae

4 Tupaia glis 2 1 1 4 3 2 6 7 36-37

PRIMATE

Cercopithecidae

5 Macaca fascicularis S S S S S S - - -

6 Trachypithecus obscurus S S - - -

RODENTIA

Sciuridae

7 Callosciurus notatus S - 1 1 2 1 1 4 3 7 38-40

8 C. caniceps 1 2 1 4 1 4 5 41

Muridae

9 Rattus tiomanicus viclana 12 12 5 2 6 6 21 42-43

10 Rattus exulans 2 6 2 3 2 8 7 44-45

11 Leopoldamys sabanus 4 6 2 3 7 8 46-47

12 Niviventer cremoriventer 3 2 1 4 48-49

13 Maxomys surifer 1 1 2 4 4 4 51-

Hystricidae

14 Hystrix brachyura S - 1 S - 1 2 -

CARNIVORA

Viveridae

15 Paradoxurus hermaphroditus S - 1 S - 2 S - 1 1 S - 1 1 2 5 R

Mustelidae

16 Lutragale perspicillata S - 2 S - 2 4 -

ARCTIODACTYLA

Suidae

17 Sus scrofa S S S + Tr S + Tr S - 3 4 -

Tragulidae

18 Tragulus kancil/napu S - 2 S - 1 S - 1 S - 2 6 -

Jostt vol 6.indd 29 7/22/10 10:09:06 PM

29

Journal of Science and Technology in the Tropics (2010) 6: 23-30


30

Journal of Science and Technology in the Tropics (2010) 6: 23-30

CHIROPTERA

Pteropodidae

19 Rousettus amplexicaudatus 1 4 3 2 52

20 Cynopterus brachyotis 2 5 3 8 5 3 14 12 53-54

21 C. horsfieldii 1 3 2 4 2 55

22 Eonycteris spelaea 1 1 3 6 4 2 8 9 56-57

23 Penthetor lucasi 2 4 4 2 4 8 58

24 Macroglossus minimus 2 2 4 2 5 5 59

25 Balionycteris maculata 2 2 3 1 60

Emballonuridae

26 Emballonura monticola 1 3 3 3 4 61

27 Taphozous melanopogon 1 2 2 1 62

Nycteridae

28 Nycteris tragata 2 2 2 2 4 63

Megadermatidae

29 Megaderma spasma 3 2 1 64

30 M. lyra 1 1 65

Rhinolophoridae

31 Rhinolophus affinis 12 8 13 11 20 24 66-67

32 R. trifoliatus 3 2 2 3 68-69

33 R. coelophyllus 3 2 3 5 3 70-71

34 R. pusillus 3 1 2 2 72-73

35 R. lepidus 1 3 2 3 3 74-75

Hipposideridae

36 Hipposideros bicolor 11 12 9 6 17 21 76-78

37 H. galeritus 3 10 14 16 11 79-80

38 H. cineraceus 2 1 1 81

39 H. diadema 3 1 2 82

40 H. larvatus 2 5 2 4 5 83-84

Vespertilionidae

41 Myotis muricolor 3 2 1 85

42 M. hasseltii 1 3 3 1 86-87

43 Murina suillus 1 2 1 3 1 88-89

44 Kerivoula papillosa 3 2 1 90

45 K. intermedia 2 4 2 4 93

46 Scotophilus kuhli 2 4 2 4 94

Molossidae

47 Tadarida mops 2 3 2 3 95-96

Total no. species 8 21 28 36 38 12

Total No. Individuals 12 64 86 101 112 30

Jostt vol 6.indd 30 7/22/10 10:09:07 PM


Journal of Science and Technology in the Tropics (2010) 6: 31-38

Antiproliferative and antioxidant properties of leaf extracts of Pereskia bleo

(Cactaceae) and their ability to limit natural and oxidant-induced

apoptotic cell death

Hui Meng Er * , Hong Lim Lee, Ammu Kutty Radhakrishnan

Faculty of Medicine and Health, International Medical University, No.126 Jalan 19/155B, Bukit Jalil,

57000 Kuala Lumpur, Malaysia

( * Corresponding author E-mail: huimeng_er@imu.edu.my)

Abstract The anti-proliferative activities of methanol extract of leaves of Pereskia bleo and its ethyl acetate,

t-butanol and aqueous fractions were studied in normal mouse fibroblast cells (NIH/3T3) and mouse

mammary cancer cells (4T1). Although the extracts did not show significant anti-proliferative effect, they

exhibited selectivity in inhibiting the proliferation of the cancer cells (4T1) compared to the normal cells (3T3).

The antioxidant properties of the leaf extracts and their ability to limit natural and oxidant-induced cell death

were also studied. Among the extracts, the t-butanol extract possessed the highest antioxidant property. The

extracts were also able to limit natural and oxidant-induced cell death in normal mouse fibroblast cells.

Keywords Pereskia bleo leaf extracts – anti-proliferative – antioxidant – apoptotic cell death

INTRODUCTION

Pereskia bleo (Kunth) DC (Cactaceae) is commonly

consumed by some ethnic groups in Malaysia for its

medicinal properties. It is claimed to have antidiabetic

and antihypertensive properties [1]. Besides, it is

widely believed by the local community that the

drink prepared by boiling the plant leaves in water is

effective in preventing and treating cancers. Hence,

the plant is commonly cultivated in the gardens.

However, there are very little in vitro and in vivo

studies to support most of these claims.

It has been shown previously that the methanol

extract of P. bleo can kill T47-D human mammary

cancer cells [2], suggesting that this plant has the

potential to be developed as a candidate for treatment

of cancers. However, in another study, it was reported

that the methanol and aqueous extracts of the leaves

did not possess any significant anti-proliferative

activity on mouse mammary cancer cells (4T1) and

normal mouse fibroblast cells (3T3) [3]. Moreover,

it was also reported that the aqueous extracts of the

leaves could form mutagenic compounds when these

are metabolized by liver enzymes [3].

Although the methanol extract and its ethyl-acetate

fraction has been reported to have high cytotoxic effect

on human nasopharyngeal epidermoid carcinoma

(KB) cells [4], their cytotoxic activity in other cell

lines such as human cervical carcinoma cell line

(CasKi), human colon carcinoma cell line (HCT116),

hormone-dependent breast carcionoma cell line

(MCF-7), and non-cancer human fibroblast cell line

(MRC-5) was less significant [4]. In another study

it was reported that the hexane, dichroloromethane,

ethyl acetate and methanol extracts of the leaves were

also non-effective in inducing cell death towards

MCF-7 (human breast cancer), HT-29 (human colon

carcinoma) and CEM-SS (human T4-lymphoblastoid)

cell lines [5].

Anti-proliferative studies carried out on the

methanol extract of the stem and its ethyl acetate,

t-butanol and aqueous fractions on mouse mammary

cancer cells (4T1) and normal mouse fibroblast cells

(NIH/3T3) did not indicate significant antiproliferative

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31

Journal of Science and Technology in the Tropics (2009) 5: 117-123


32

Journal of Science and Technology in the Tropics (2010) 6: 31-38

activity [6]. A detailed analysis on the methodologies

employed in the previous studies suggested that

the inconsistent findings on the anti-proliferative

activity could be attributed to differences in the parts

of the plant studied (e.g. leaf, stem, whole plant),

plant extraction procedure, cell lines used and antiproliferation

assay conditions [3].

The diversity of secondary metabolites in plants

is well known. They play a major role in contributing

to the plants’ natural defense mechanism against

predators or diseases caused by microorganisms. As

the stems of P. bleo are heavily covered with sharp

spines that can protect the plant against preys, they

may also be associated with high level of secondary

metabolites of important medicinal properties, such

as anticancer activity. However, previous study has

shown that the stem extracts do not possess significant

anti-proliferative activity against NIH/3T3 and 4T1

cells [6]. In the current study, the same procedure

was employed for the extraction and fractionation of

the leaves of the plant using the same solvents, and

the anti-proliferative activities of the extracts were

assessed using the same cell lines.

The mechanism of cell death caused by oxidative

stress has been studied widely [7]. The generation of

endogenous reactive oxygen or nitrogen species as

a result of oxidative stress can lead to tissue injury

implicated in many diseases. Many antioxidant

compounds are found in higher plants and they have

been shown to possess the ability to quench free

radicals and reactive oxygen species in vitro [8]. In

this study, the in vitro antioxidant property of the leaf

extracts, as well as their ability to limit natural and

oxidant-induced cell death were evaluated.

MATERIALS AND METHODS

Plant Material

The plant cuttings of P. bleo were collected from Taman

Pertanian (Agricultural Garden) of the Universiti

Putra Malaysia (UPM), Selangor, Malaysia. The

voucher specimen (No. ACP 0116) was deposited in

the herbarium of Institut Biosains of UPM.

Preparation of leaf extracts

The leaves of P. bleo were separated, cleaned with

water and dried in a convection oven at 40 o C until

consistent weights were obtained. The dried leaves

were blended into powder form using an electric

blender. A weighed amount (20 g) of the leaf powder

was extracted with methanol (250 mL) using a soxhlet

extractor for four hours. The process was repeated

five times in order to obtain sufficient amount of

the extracts for further testing and the extracts were

combined. The methanol solvent in the combined

extract was removed under reduced pressure at

40 o C using a rotary evaporator and the extract was

further dried using a vacuum concentrator at 40 o C

until a consistent weight was obtained. A portion of

the methanol extract was fractionated by partitioning

into ethyl acetate, t-butanol and water. The solvents

in these fractionated extracts were removed under

reduced pressure at 40 o C using a rotary evaporator.

The ethyl acetate and t-butanol extracts were further

dried to consistent weight using a vacuum concentrator

at 40 o C. The aqueous extract was freeze-dried to a

consistent weight. The yields of the methanol, ethyl

acetate, t-butanol and aqueous extracts were 7.7%,

0.2%, 3.8% and 7.1%, respectively, based on the dry

weight of the leaves.

ABTS radical cation scavenging activity of

P. bleo leaf extracts

The assay is based on the ability of substances with

antioxidant properties to scavenge 2,2’-azinobis-

(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)

radical cations, which are blue-green in colour and

absorb at 734 nm [9]. Any positive reaction will lead

to the decolourisation of the ABTS radical cations.

The methanol extracts of the leaves were dissolved

in 0.01 M phosphate buffered saline (PBS) while the

ethyl acetate and t-butanol extracts were dissolved

in 0.8% (v/v) DMSO. Trolox and vitamin C were

used as positive controls. To perform this assay, the

ABTS solution (715 µL, 5 mM) was mixed with

potassium persulphate solution (285 µL, 2.5 mM)

and the reaction mixture was kept in the dark at room

temperature for 12-16 hours for the generation of

ABTS radical cations.

The ABTS radical cation solution was

appropriately diluted with 0.01 M PBS to give an

absorbance of 0.7 ± 0.2 at 734 nm. To 990 µL of this

diluted ABTS radical cation solution, 10 µL of each

of the concentrations of the leaf extracts, Trolox or

vitamin C was added. The absorbance of the mixture

at 734 nm was recorded at every minute for a period

of 12 minutes. The experiment was repeated five

times for each concentration of the extracts (2.5

to 50 µg/mL for t-butanol extract; 5 to 100 µg/mL

for the other extracts), Trolox (0.25 to 2.5 mM) or

Jostt vol 6.indd 32 7/22/10 10:09:08 PM


vitamin C (0.25 to 2.5 mM). Lower concentrations

of the t-butanol extract were used compared to the

other extracts due to the difficulty in dissolving this

extract. The percentage inhibition of ABTS radical

cation at each time point was calculated using the

formula: Percentage inhibition = [(Initial absorbance

- Absorbance at time of interest) / Initial absorbance]

x 100%.

Cell lines

The NIH/3T3 (normal mouse fibroblast cell line)

and 4T1 (mouse mammary cancer cell line) cells

were purchased from the American Type Culture

Collection (ATCC, Rockville). They were cultured

in RPMI 1640 medium containing L-glutamine and

supplemented with 10% FBS, 1% HEPES buffer

solution, 1% sodium pyruvate (100 mM) and 0.5%

penicillin-streptomycin in a humidified 5% CO 2

incubator at 37 o C.

Anti-proliferation assay

Cell proliferation was analysed using the MTT

assay [10]. This assay is based on the ability

of the mitochondria of living cells to reduce

a chemical, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium

bromide (MTT), which is a

yellow tetrazolium salt, to purple formazan product

that is insoluble in the aqueous phase. The NIH/3T3

or 4T1 cells (100 µL of 5x10 4 cells) were cultured in

a sterile 96-well flat-bottom tissue culture plate. The

plate was incubated at 37 o C in a humidified 5% CO 2

incubator for two hours. The methanol, ethyl acetate,

t-butanol and aqueous extracts of the leaves were

appropriately diluted (50 to 800 µg/mL) in medium

containing 0.8% (v/v) dimethyl sulphoxide (DMSO).

Various concentrations (25 to 100 µg/mL) of cisplatin,

which was used as a positive control was also prepared

in culture medium containing 0.8% (v/v) DMSO. The

diluted solutions were filtered using sterile 0.22 µm

filter units. Following the two hour incubation, 100

µL of the diluted leaf extracts or cisplatin were added

to the 96-well culture plates containing the NIH/3T3

or 4T1 cells (in triplicates). The final concentrations

of the leaf extracts in this assay ranged from 12.5 to

400 µg/mL while cisplatin ranged from 1.6 to 50 µg/

mL. Cells cultured in medium containing 0.4 % (v/v)

DMSO were used as negative control. The plates

were incubated at 37 o C in a humidified CO 2 incubator

for 72 hours. Then MTT solution (20 µL, 5 mg/mL in

PBS) was added to each of the wells and the plate was

returned to the incubator for 4 hours. Following this,

the supernatant in each well was removed carefully

(the purple formazan product was attached to the

bottom of the well). Dimethylsulphoxide (50 µL)

was then added to each well to dissolve the purple

formazan product. The absorbance of the solution

in each well was determined using an ELISA plate

reader at 570 nm. The percentage cell viability in each

well was calculated by the formula: Percentage cell

viability = (Absorbance at 570 nm of treated well /

Absorbance at 570 nm of negative control) × 100%.

Cell death detection by enzyme-linked

immunosorbent assay (ELISA)

The NIH/3T3 cells were diluted in culture medium

to a concentration of 1 x 10 5 cells/mL. This diluted

cell suspension (50 µL) was added to the wells of a

sterile 96-well flat-bottom tissue culture plate. The

plate was left in a humidified 5% CO 2 incubator at

37 o C for two hours. The extracts from the leaves

(methanol, ethyl acetate, t-butanol and aqueous

extracts) were dissolved in DMSO and diluted with

the culture medium to a concentration of 100 and 200

µg/mL. The concentration of DMSO in the diluted

solutions was 0.8% (v/v). These solutions were

filtered using a sterile 0.22 µm filter unit. A 50 µL

aliquot of the diluted solution (200 or 100 µg/mL)

of the leaf extracts, Trolox or vitamin C was then

added (in triplicates) to the respective wells in the

plate containing NIH/3T3 cells at the concentration

of 5 x 10 3 cells per well. To test for the ability of

these extracts to protect NIH/3T3 cells against DPPH

(2,2-diphenyl-1-Picrylhydrazyl) induced cell death,

50 µL of 30 µM DPPH solution was added to the

relevant wells one hour after the NIH/3T3 cells were

incubated in the presence of the appropriately diluted

extracts, Trolox, Vitamin C or cultured medium. The

plate was incubated at 37 o C for 24 hours. Trolox and

vitamin C were used as the positive controls while

medium containing 0.4% (v/v) DMSO served as the

negative control.

Cell death due to apoptosis was performed using

the Cell Death Detection ELISA PLUS kit according to

the manufacturer’s instruction as described previously

[3]. This assay is based on a quantitative sandwichenzyme-immunoassay

principle that utilises mouse

monoclonal antibodies directed against DNA and

histones associated DNA fragments. Briefly, the plate

was centrifuged at 200 × g for 10 minutes at room

temperature after 24 hours. The supernatant was

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Journal of Science and Technology in the Tropics (2010) 6: 31-38


34

Journal of Science and Technology in the Tropics (2010) 6: 31-38

completely removed. Then, the pellet in each well

was resuspended in 200 µL of lysis buffer (provided

by manufacturer) and the plate was incubated at

room temperature for 30 minutes. Following this,

the plate was centrifuged at 200 × g for 5 minutes.

An aliquot of 20 µL of the supernatant containing

the cytoplasmic fraction in each well was carefully

transferred into appropriate wells of a streptavidincoated

plate (provided by manufacturer) for further

analysis. Then, 80 µL of an immunoreagent (provided

by manufacturer) was added into each well. The plate

was covered with an adhesive foil and gently shaken

at 200 rpm for two hours. The solution in each well

was then removed and the wells were thoroughly

rinsed thrice with 300 µL of incubation buffer

(provided by manufacturer). Following this, 100 µL

of ABTS solution (provided by manufacturer) was

added into each well and the plate was gently shaken

at 200 rpm until suitable colour development was

obtained (20-30 minutes). The absorbance of each

well was determined using an ELISA plate reader at

405 nm with a reference wavelength of 490 nm. The

enrichment factor was calculated using the following

formula provided by the manufacturer: Enrichment

factor = (Absorbance at 405 nm of treated cells) /

(Absorbance at 405 nm of untreated cells).

RESULTS

ABTS radical cation scavenging activity of

P. bleo leaf extracts

The percentage inhibition of the ABTS radical

cations over 12 minutes by the leaf extracts at the

highest concentrations is shown in Figure 1. Figure

2 shows the percentage inhibition of ABTS radical

cations at the sixth minute versus concentrations

of the leaf extracts, as well as the linear regression

equations for the graphs of the leaf extracts. The

Trolox Equivalent (TE) per gram dry extract for the

leaf extracts are presented in Table 1. Among the

leaf extracts, the t-butanol leaf extract exhibited the

Table 1. Trolox Equivalent (TE) µmole

per gram dry extract of Pereskia bleo leaf

extract and vitamin C.

Extract TE ± S.D.

Methanol leaf extract 176 ± 2

Ethyl acetate leaf extract 208 ± 3

t-Butanol leaf extract 416 ± 15

Aqueous leaf extract 181 ± 2

Vitamin C 5158 ± 3

highest ABTS radical cation scavenging activity and

this was followed by the ethyl acetate leaf extract.

The methanol leaf extract and aqueous leaf extract

showed comparable ABTS radical cation scavenging

activity and were the lowest among the leaf extracts.

Anti-proliferation assay

There was a gradual decrease in cell viability with

increasing concentrations of all the leaf extracts of

Percentage inhibition (%)

Percentage inhibition of

ABTS radical cation (%)

100.0

90.0

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (Min)

Figure 1. Percentage inhibition of ABTS radical

cations in the presence of the extracts from the

leaves of Pereskia bleo (100 µg/mL of methanol,

ethyl acetate or aqueous extracts, 50 µg/mL of

t-butanol extract) over 12 minutes. The data are

expressed as the mean percentages inhibition of

ABTS radical cations ± standard deviation (S.D.).

denotes methanol extract at 100 µg/mL; ∆

denotes ethyl acetate extract at 100 µg/mL; ×

denotes t-butanol extract at 50 µg/mL; ◊ denotes

aqueous extract at 100 µg/mL.

100

90

80

70

60

50

40

30

20

10

0

0 20 40 60 80 100 120

Concentration (ug/mL)

Figure 2. Percentage inhibition of ABTS radical

cations by various concentrations of the extracts

from the leaves of Pereskia bleo at the sixth minute.

denotes methanol extract; ∆ denotes ethyl acetate

extract; × denotes t-butanol extract; ◊ denotes aqueous

extract. The linear regression equations for methanol

extract: y = 0.6668x + 8.9156; ethyl acetate extract: y

= 0.7867x + 0.9745; t-butanol extract: y = 1.5734x +

0.9745; aqueous extract: y = 0.683x + 14.789.

Jostt vol 6.indd 34 7/22/10 10:09:11 PM


P. bleo against both the 4T1 and 3T3 cells (Fig. 3).

However, the IC 50 values for all the extracts were

greater than the highest tested concentration (400

µg/mL). We could not use concentrations higher

than 400 µg/mL due to insolubility of these extracts

at high concentrations. The percentage cell viability

values of the 4T1 and 3T3 cells treated with 400

µg/mL of the various leaf extracts for 72 hours are

presented in Table 2. All the extracts resulted in a

greater reduction of the percentage viability of the

4T1 cells compared to the 3T3 cells.

Table 2. The percentage of viable 4T1 and 3T3 cultured

cells treated with 400 µg/mL of extract from the leaves of

Pereskia bleo for 72 hours.

Extract

% ± S.D. of viable cells

4T1 cells 3T3 cells

Methanol leaf extract 80.81 ± 0.97 107.55 ± 2.00

Ethyl acetate stem extract 72.63 ± 1.65 73.36 ± 7.76

t-Butanol leaf extract 78.55 ± 4.92 100.37 ± 3.57

Aqueous leaf extract 77.44 ± 2.10 105.89 ± 5.41

Figure 3. Percentage of viable (a) 4T1 cells and (b) 3T3

cells cultured in the presence of the methanol, ethyl

acetate, t-butanol and aqueous extracts of the leaves of

Pereskia bleo for 72 hours.The data are expressed as the

mean percentages of cell viability ± standard deviation

(S.D.). denotes methanol extract; ∆ denotes ethyl

acetate extract; × denotes t-butanol extract; ◊ denotes

aqueous extract; + denotes cisplatin.

Ability of leaf extracts of P. bleo to limit natural

apoptotic cell death in 3T3 cells

Figure 4 shows the enrichment factors calculated

when 3T3 cells were cultured in the presence and

absence of 50 µg/mL or 100 µg/mL extracts from the

leaves of P. bleo. The enrichment factor for untreated

cells (i.e. negative control) was 1.0. Enrichment

factor value less than 1.0 indicated a reduction in cell

death by apoptosis. All the leaf extracts were able to

reduce natural apoptotic cell death in 3T3 cells. The

ability to limit natural apoptotic cell death in 3T3

cells was greater at 50 µg/mL than at 100 µg/mL for

all extracts.

Ability of leaf extracts of P. bleo to limit oxidantinduced

apoptotic cell death in 3T3 cells

An increase in apoptotic cell death was observed in

the 3T3 cells cultured for 24 hours in the presence

of 10 µM DPPH (Fig. 5). However, the extent of

the DPPH-induced apoptotic cell death was reduced

when the cells were pre-incubated with extracts from

the leaves for one hour before DPPH were added to

the culture wells. There was no significant difference

in the level of oxidant-induced apoptotic cell death

when the 3T3 cells were cultured in the presence of

50 µg/mL of the leaf extract, compared to the higher

concentration used i.e.100 µg/mL. The protective

effects of all the extracts against oxidant-induced

cell death were greater than Trolox or vitamin C.

Figure 4. The ability of extracts from the leaves of

Pereskia bleo to limit natural apoptotic death in 3T3 cells.

The data are expressed as the mean enrichment factors

± standard deviation (S.D.). denotes cells in media

only; * denotes cells in media containing 0.4% DMSO;

◊ denotes aqueous extract; denotes methanol extract;

× denotes t-butanol extract; ∆ denotes ethyl acetate

extract; + denotes vitamin C; - denotes trolox.

Jostt vol 6.indd 35 7/22/10 10:09:15 PM


35

Journal of Science and Technology in the Tropics (2010) 6: 31-38


36

Journal of Science and Technology in the Tropics (2010) 6: 31-38

Among the extracts, the ethyl acetate extract showed

the highest protective effect against oxidant-induced

apoptotic cell death.

DISCUSSION

The sequential fractionation of the crude methanol

extracts of the leaves of P. bleo used in this study

showed that the bioactive compounds with antioxidant

activity were concentrated in the t-butanol extract

(416 µmole Trolox Equivalent per gram dry weight),

followed by the ethyl acetate extract. The methanol

and aqueous extracts had similar antioxidant

properties. These antioxidant properties are moderate

compared to those of commercial black teas and

green teas which have antioxidant capacity ranging

between 235 µmole Trolox Equivalent per gram dry

weight and 1526 µmole Trolox Equivalent per gram

dry weight [9]. The antioxidant effects of the extracts

are also much less than that of Vitamin C, a known

antioxidant compound, which has an antioxidant

capacity of 5158 Trolox Equivalent per gram dry

weight [9]. As such, the leaf of P. bleo cannot be

considered to be a rich source of antioxidants.

The IC 50 values for all the crude and fractionated

extracts of the leaves of P. bleo were greater than 400

µg/mL. The extracts could not be tested at higher

concentrations due to the lack of solubility. According

to the National Cancer Institute guideline, the crude

Figure 5. The ability of extracts from the leaves of

Pereskia bleo to limit DPPH-induced apoptotic death in

3T3 cells. The data are expressed as the mean enrichment

factors ± standard deviation (S.D.). o denotes cells in

media only; * denotes cells treated with DPPH; ◊ denotes

aqueous extract; denotes methanol extract; ×

denotes t-butanol extract; Δ denotes ethyl acetate

extract; + denotes vitamin C; - denotes trolox.

extract of a plant should have an IC 50 value of less

than 20 µg/mL in order to be considered as cytotoxic

against the treated cells [11]. The results suggested

that these extracts did not contain significant amount

of compounds with anti-proliferative properties.

Since the anti-proliferative activity of the fractionated

extracts (ethyl acetate, t-butanol and aqueous extracts)

are not greater than that of the crude methanol extract,

the methanol extract is unlikely to contain any

significant amount of anti-proliferative compounds.

A comparison with the reported anti-proliferative

activity of the stem extracts of P. bleo [6] shows

that the leaf extracts have higher anti-proliferative

activity. Moreover, the leaf extracts have a higher

anti-proliferative effect on the 4T1 mouse mammary

cancer cells compared to the normal 3T3 mouse

fibroblast cells. However, the reverse trend has been

reported for the stem extracts [6]. This indicates that

the leaf extracts of P. bleo might be a better target to

be developed as anti-cancer agent compared to the

stem extracts.

Treatment of the normal mouse fibroblast cells

(3T3) with the leaf extracts of P. bleo for 24 hours

resulted in a lower level of apoptosis in the cells

compared to that occurring in the non extracttreated

cells grown under the same conditions. The

results indicated that all the methanol, ethyl acetate,

t-butanol and aqueous extracts had the ability to

protect the 3T3 cells against natural programmed

cell death by apoptosis. The protective effects were

more obvious at 50 µg/mL compared to 100 µg/mL

of the extracts. The higher level of apoptosis at the

higher extract concentration was consistent with the

results of the anti-proliferation assay which showed

a gradual decrease in cell viability with increasing

extract concentration. All the fractionated extracts of

the leaves of P. bleo showed higher apoptosis-limiting

activities than the crude methanol extract, except

the aqueous extract. This suggested that the crude

methanol extracts contained bioactive component(s)

that could protect the normal mouse fibroblast

cells against natural apoptotic cell death, and these

components could be concentrated by fractionating

the crude methanol extracts.

The protective effects of the leaf extracts of P. bleo

against oxidant-induced cell death were also evaluated

by pre-treating the 3T3 cells with the extracts before

the oxidant, 2,2-Diphenyl-1-Picrylhydrazyl (DPPH),

was added. All the extracts were able to limit oxidantinduced

cell death, with the ethyl acetate extract being

Jostt vol 6.indd 36 7/22/10 10:09:17 PM


the most effective. The ability to limit oxidant-induced

cell death was not significantly different between 50

µg/mL and 100 µg/mL of the extracts, indicating that

the maximum effective concentration was reached.

The findings on the protective effects of the

extracts against oxidant-induced cell death were

consistent with the moderate antioxidant activities of

the extracts determined by the ABTS radical cation

scavenging assay. Nevertheless, the t-butanol extract

possessed the highest in vitro antioxidant activity,

whereas the ethyl acetate extract was more effective

in limiting oxidant-induced cell death. The ability of

the extracts to scavenge ABTS radical cation implied

that these extracts could act as reactive oxygen

species scavenger in diseases caused by oxidantinduced

cell death. An example of plants which

possess such property is Pogostemon cablin, a wellknown

Korean traditional medicine that has been

proven to be beneficial for patients with cerebral

stroke [12]. The water extract of P. cablin has been

shown to be able to protect the human neuroglioma

cells against necrotic and apoptotic cell death induced

by hydrogen peroxide. The protective effects of the

P. bleo extracts against oxidant-induced cell death

should be investigated further for their potential to

be developed for treatment of neurodegenerative

diseases which can be caused by reactive oxygen

species.

From this study, we can conclude that all of

the crude methanol and fractionated ethyl acetate,

1. Goh K.L. (2000) Malaysian Herbaceous Plants.

Millennium Edition. Advanco Press, Malaysia.

2.

3.

4.

5.

Tan M.L., Sulaiman S.F., Najimuddin N., Samian

M.R. and Tengku Muhammad T.S. (2005) Methanolic

extract of Pereskia bleo (Kunth) DC. (Cactaceae)

induces apoptosis in breast carcinoma, T47-D cell

line. Journal of Ethnopharmacology 96: 287-294.

Er H.M., Cheng E. and Radhakrishnan A.K.

(2007) Anti-proliferative and mutagenic activities

of aqueous and methanol extracts of leaves from

Pereskia bleo (Kunth) DC (Cactaceae). Journal of

Ethnopharmacology 113: 448-456.

Abd Malek S.N., Abdul Wahab N., Yaacob H., Sim

K.S., Hong S.L., Lee G.S. and Rahman S.N.S.A.

(2008) Cytotoxic activity of Pereskia bleo (Cactaceae)

against selected human cell lines. International

Journal of Cancer Research 4: 20-27.

Wahab S.I.A., Abdul A.B., Mohan S.M., Al-Zubairi

A.S., Elhassan M.M. and Ibrahim M.Y. (2009)

REFERENCES

t-butanol and aqueous extracts of the leaves of P.

bleo do not have significant anti-proliferative effect

on both the mouse mammary cancer cells (4T1) and

the normal mouse fibroblast cells (3T3). They are not

cytotoxic, based on the criterion that a crude extract

should have an IC 50 of less than 20 µg/mL for it to

be considered cytotoxic against the treated cells [11].

Nevertheless, the leaf extracts exhibited selectivity in

inhibiting the proliferation of the mouse mammary

cancer cells as opposed to the normal mouse fibroblast

cells. One possible mechanism for the selective killing

of the cancer cells may be related to the ability of the

extracts to activate the p53 function in tumour cells,

causing their growth arrest or apoptosis, as reported

in Ashwagandha (Withania somnifera), a herb

commonly used in Ayurvedic medicine [13]. Among

the extracts tested, the t-butanol extract of the leaves

of P. bleo possesses the highest antioxidant property.

Yet, the antioxidant capacity is only moderate when

compared to other known antioxidant substances

such as green tea leaves and vitamin C. The ability of

the extracts of the leaves of P. bleo in limiting natural

and oxidant-induced cell death in normal mouse

fibroblast cells suggests that the plant may be useful

as a remedy for diseases related to oxidative stress.

Acknowledgement – We would like to thank Taman

Pertanian Malaysia, Universiti Putra Malaysia, for kind

donation of the Pereskia bleo plant. This work was supported

by a grant from the International Medical University.

6.

7.

8.

9.

Biological activities of Pereskia bleo extracts.

International Journal of Pharmacology 5: 71-75.

Lee H.L., Er H.M. and Radhakrishnan A.K. (2009)

In vitro anti-proliferative and antioxidant activities of

stem extracts of Pereskia bleo (Kunth) DC (Cactaceae).

Malaysian Journal of Science 28: 225-239.

Ryter S.W., Kim H.P., Hoetzel A., Park J.W., Nakahira

k., Wang X. and Choi A.M.K. (2007) Mechanisms of

cell death in oxidative stress. Antioxidants and Redox

Signaling 9: 49-89.

Larson R.A. (1988) The antioxidants of higher plants.

Phytochemistry 27: 969-978.

Re R., Pellegrini N., Proteggente A., Pannala A.,

Yang M. and Rice-Evans C. (1999) Antioxidant

activity applying an improved ABTS radical cation

decolorization assay. Free Radical Biology and

Medicine 26: 1231-1237.

Carmichael J., DeGraff W.G., Gazdar A.F., Minna J.D.

and Mitchell J.B. (1987) Evaluation of a tetrazolium-

Jostt vol 6.indd 37 7/22/10 10:09:17 PM

10.

37

Journal of Science and Technology in the Tropics (2010) 6: 31-38


38

Journal of Science and Technology in the Tropics (2010) 6: 31-38

11.

12.

based semiautomated colorimetric assay: assessment

of radiosensitivity. Cancer Research 47: 943-946.

Geran R.I., Greenberg N.H., Macdonald M.M.,

Schumacher A.M. and Abbott B.J. (1972) Protocols

for screening chemical agents and natural products

against animal tumours and other biological system.

Cancer Chemotherapy Reports 3: 59-61.

Kim H.W., Cho S.J., Kim B., Cho S.I. and Kim Y.K.

(2010) Pogostemon cablin as ROS scavenger in

13.

oxidant-induced cell death of human neuroglioma

cells. Evidence-based Complementary and Alternative

Medicine 7: 239-247.

Widodo N., Kaur K., Shrestha B.G., Takagi Y., Ishii

T., Whadhwa R. and Kaul S.C. (2007) Selective

killing of cancer cells by leaf extract of Asgwagandga:

Identification of a tumor-inhibitory factor and the

first molecular insights to its effect. Clinical Cancer

Research 13: 2298-2306.

Jostt vol 6.indd 38 7/22/10 10:09:18 PM


Journal of Science and Technology in the Tropics (2010) 6: 39-42

Abdominal colour polymorphism in female Asian Golden Web Spider

Nephila antipodiana (Araneae: Nephilidae)

Yong Hoi Sen1 , Rosli Hashim1 , Daicus Belabut1 and Lim Phaik Eem1,2 1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

2Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

(E-mail: yong@um.edu.my)

Abstract Nephila antipodiana (Batik Golden Web Spider) occurred in large numbers in several localities in

Kelantan and Terengganu, east coast of Peninsular Malaysia. Three abdominal (opisthosomal) colour morphs

– yellow, greenish-yellow and reddish-brown – were present in adult female spiders. Only two colour morphs

appear to be present in a particular locality. The yellow morph occurred in all the localities investigated. Of

the two non-yellow morphs, the reddish-brown morph was found only in one locality while the greenish-yellow

morph was more widespread. The present study does not show unequivocally the association of colouration

with habitat usage. Whether yellow colour in N. antipodiana confers a selective advantage remains to be

verified. In addition to opisthosomal colour polymorphism, the colour and number of spots (or sigillae) on the

dorsal surface of the abdomen of female N. antipodiana are also variable. The juvenile spiders have different

colour pattern from the adults.

Keywords polymorphism – golden web spider – Arachnida – Malaysia – opisthosoma colour

INTRODUCTION

The Batik (or Asian) Golden Web Spider Nephila

antipodiana (Walckenaer 1842) was first reported

in Peninsular Malaysia (and Malaysia) in 2009 [1].

It was originally described as Epeira antipodiana

Walckenaer 1842. It is distributed in China, Philippines

to New Guinea, Solomon Is., and Queensland [2]. In

Southeast Asia it has been recorded in Singapore,

Indonesia, Thailand and the Philippines [3, 4]. It has

also been found in Borneo (Joseph K.H. Koh, pers.

comm.).

It is evident from the literature that N. antipodiana

is a variable species [1, 3-5]. In the Philippines the

dorsal colour of the abdomen (opisthosoma) in the

female is yellow, with six pairs of subovate yellow

spots dorsally arranged longitudinally in rows, each

spot with a thin black margin [4]. The abdomen of

the female spider in Australasia is pale yellow, with

darker markings on anterior and posterior margins,

and without large yellow spots [5]. In Singapore the

dorsal colour of the abdomen may be yellowish green

[3] or reddish brown [1] with yellow spots.

Although abdominal (opisthosomal) colour

variation occurs in the species, as evidenced in

the different colour morphs, there is no report on

polymorphism. We report here the occurrence of

polymorphism in the dorsal abdominal colour of

adult female N. antipodiana in Peninsular Malaysia.

MATERIALS AND METHODS

A field survey of N. antipodiana was carried out

from 12-15 February 2010 in Kelantan and

Terengganu, Peninsular Malaysia. The dorsal colour

(yellow, greenish-yellow, and reddish-brown) of the

abdomen of every adult female spider in a chosen

locality was recorded. The number of juvenile and

associated male individuals was also recorded.

Five localities were studied – four in Kelantan

(Pantai Melawi and Sungai Dua in Bachok, Tok

Bok in Machang, and Gua Musang highway) and

one in Terengganu (Lata Belatan). Pantai Melawi

consisted of eight patches which were separated

although not far from each other.

Bachok is situated near the coast, with typical

Malay village setting. Both Machang and Gua

Musang are in the interior. Lata Belatan is a forest

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39

Journal of Science and Technology in the Tropics (2009) 5: 125-131


40

Journal of Science and Technology in the Tropics (2010) 6: 39-42

recreation park – the study was carried out along the

road bordered by plantation.

Representative specimens were collected and

preserved in ethanol. Photographs were also taken of

the various colour morphs and life stages.

RESULTS

Three principal abdominal colour morphs – yellow

(Figs. 1, 2), greenish yellow (Fig. 3) and reddish

brown (Fig. 4) – were observed in the adult female

N. antipodiana. Their occurrence and abundance in

four localities in Kelantan and one in Terengganu are

summarized in Table 1. The juveniles (Fig. 5) and

males were not included as the former had different

colour pattern and the male did not show colour

variation.

The yellow morph was present in all the five

localities. Of the other two morphs, the greenishyellow

morph was found only in Kelantan while the

reddish-brown morph was observed in Lata Belatan,

Terengganu. The frequency of the yellow morph

ranged from 7.69% in Sungai Dua, Bachok to 25.71%

in Lata Belatan, Terengganu (Table 1).

DISCUSSION

Colour and pattern variation in spiders has been

widely investigated [6]. To-date three major classes

of pigments – ommochromes (yellow, red, brown),

bilins (blue, green) and guanine (white, silver) – have

been identified. The earlier work before 1998 on the

evolution and ecology of spider colouration has been

well reviewed [6]. Although polymorphisms have

been reported for a number of species, the genetic

bases have generally not been elucidated. In general,

sex linkage appears to be absent but sex limitation

is quite common. It is acknowledged that the

manifestation of spider colouration is very complex,

involving a host of possible factors. Various functions

have been suggested – cryptic/disruptic, mimetic and

aposematic as well as thermoregulatory.

The Hawaiian happy-face spider, Theridion

grallator Simon (Therididae) provided the most

spectacular example of colour polymorphism in

spiders. In this spider, the opisthosomal colour may

be controlled by simple Mendelian alleles at a single

autosomal locus or multiple closely linked loci.

The plain yellow morph is recessive to the other

colour morphs [7, 8]. More recently, another species

Theridion californicum Banks has been found to

exhibit an equally extraordinary visible colour

and pattern polymorphism [9]. The polymorphism

comprises one common morph (Yellow) and at least

ten relatively rare patterned morphs, with Yellow

probably recessive to all other morphs.

In the present study, the yellow morph of female N.

antipodiana occurred in all the localities investigated

(Table 1). Lata Belatan had the highest percentage

(25.71%). This locality had denser vegetation, being

in a plantation area. In Gua Musang highway, an

open area, the percentage was 16.98%. Although it

has been suggested that yellow colour is most cryptic

in the below-leaf environment and hence selected for

Table 1. Abundance of abdominal colour morphs in adult female Nephila antipodiana from

four localities in Kelantan and Terengganu, east coast of Peninsular Malaysia, recorded in

February 2010.

Locality Greenish Yellow Yellow Reddish Brown % Yellow

Bachok: Pantai Melawi

1 outside chalet

2 coconut palms

3 house compound

4 waste land

5 coconut palms

6 Hibiscus tiliaceus

7 coconut palms

8 open space

Total (1 – 8)

2

28

39

18

26

33

8

7

161

2

3

1

5

6

2

19

50.00

9.68

0

0

3.70

13.16

42.86

22.22

10.56

Bachok: Sungai Dua 24 2 7.69

Machang: Tok Bok 82 10 10.87

Gua Musang: highway 44 9 16.98

Terengganu: Lata Belatan 27 78 25.71

Jostt vol 6.indd 40 7/22/10 10:09:19 PM


Figures 1-4. Abdominal colour morphs of adult female Nephila antipodiana (photos: H.S. Yong).

1. Yellow morph; 2. Venter of a Yellow morph; 3. Greenish-yellow morph; 4. Reddish-brown morph.

[9], the present study does not show unequivocally

the association of colouration with habitat usage.

Whether yellow colour in N. antipodiana confers

a selective advantage remains to be verified. In

Pantai Melawi with a mixture of micro-habitats, the

percentage of yellow morph ranged from 9.68% to

42.86% in patches with coconut palms while in an

open space it was 22.22% in contrat to 13.16% in an

area with Hibiscus tiliaceus (Table 1).

It is noteworthy that in the present study only two

colour morphs appear to be present in a particular

locality (Table 1). Of the two non-yellow morphs, the

reddish-brown morph was found only in one locality

(Lata Belatan) whereas the greenish-yellow morph

Jostt vol 6.indd 41 7/22/10 10:09:20 PM

41

Journal of Science and Technology in the Tropics (2010) 6: 39-42


42

Journal of Science and Technology in the Tropics (2010) 6: 39-42

was more widespread. The reddish-brown morph

was found in Teluk Chempedak, Pahang where N.

antipodiana was first reported for Peninsular Malaysia

[1]. In this locality, a yellow individual had been

observed in the hill forest (H. S. Yong, unpublished

information). At the University of Malaya, a single

individual of the greenish morph had been found

among ornamental palms (H. S. Yong, personal

observation). The significance of the presence of only

a single non-yellow morph in a particular locality

remains to be elucidated.

In addition to opisthosomal colour polymorphism,

the colour and number of spots (or sigillae) on the

dorsal surface of the abdomen of N. antipodiana are

also variable. The dorsum may be marked with spots

or sigillae. In the Philippines, the yellow morph has

1. Yong H.S. (2009) Nephila antipodiana (Araneae:

Nephilidae) from Pahang: a new record for

Peninsular Malaysia. Journal of Science and

Technology in the Tropics 5: 19-21.

2. Platnick N.I. (2010) The world spider catalog, version

10.5. American Museum of Natural History, online

at http://research.amnh.org/entomology/spiders

/catalog/index.html.

3. Koh J.K.H. (1989) A guide to common Singapore

spiders. Singapore Science Centre, Singapore.

4. Barrion A.T. and Litsinger J.A. (1995) Riceland spiders

5.

6.


 


Figure 5. Juvenile female Nephila antipodiana with

different colour pattern from adult female spider. (photo:

H.S. Yong)

of South and Southeast Asia. CAB International,

Wallingford, UK.

Harvey M.S., Austin A.D. and Adams M. (2007) The

systematic and biology of the spider genus Nephila

(Araneae: Nephilidae) in the Australasian region.

Invertebrate Systematics 21: 407-451.

Oxford G.S. and Gillespie R.G. (1998) Evolution

REFERENCES

six pairs of subovate yellow spots, each with a thin

black margin [4]. In Australasia, the dorsal surface

has four pairs of sigillae [5]. The yellow morph in the

present study (Peninsular Malaysia) is characterized

by five pairs of yellow spots/sigillae, each well

defined by black margin. There may also be a sigilla/

spot situated at the middle of the dorsal surface (Fig.

1).

The yellow morph of N. antipodiana in general

appears to possess fewer spots than the non-yellow

morphs. In the present study, the yellow morph had

five pairs of spots/sigillae. On the other hand, the

greenish-yellow and reddish-brown morphs had nine

pairs of spots which were variable in size. Another

difference is the spots in the yellow morph tended

to assume the appearance of sigillae – depression at

least at the posterior part of the spot. The intensity

and dimension of the black outline are also variable.

Spots in the greenish-yellow and reddish-brown

morphs are not marked by black outline.

In sum, N. antipodiana is highly variable

in colouration and pattern/marking. Cursory

observations indicate the presence of other colour

morphs. Detailed studies are needed to elucidate the

ontogeny of colour and pattern development, and

the function and mechanism of the variation and

polymorphism. N. antipodiana could be an excellent

model for evolutionary and ecological studies.

Acknowledgements – We thank the University of

Malaya for financial and other supports to carry out this

study.

7.

8.

9.

and ecology of spider coloration. Annual Review of

Entomology 43: 619-643.

Gillespie R.G. and Tabashnik B. (1989) What makes

a happy face? Determinants of colour pattern in the

spider Theridion grallator (Araneae, Theridiidae).

Heredity 62: 335-363.

Oxford G.S. and Gillespie R.G. (1996) Genetics of a

colour polymorphism in Theridion grallator (Araneae:

Theridiidae), the Hawaiian happy-face spider, from

Greater Maui. Heredity 76: 238-248.

Oxford G.S. (2009) An exuberant, undescribed

colour polymorphism in Theridion californicum

(Araneae, Theridiidae): implications for a theridiid

pattern ground plan and the convergent evolution of

visible morphs. Biological Journal of the Linnean

Society 96: 23-34. With 2 figures.

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Journal of Science and Technology in the Tropics (2010) 6: 43-47

Scaled down operation of the United Nations University/International Centre

for Theoretical Physics Plasma Focus Facility (UNU/ICTP PFF) as an

extreme ultraviolet source

Rattachat Mongkolnavin11* 1 2

, Prajya Tangitsomboon and Chiow San Wong

1Department of Physics, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

2Plasma Research Laboratory, Physics Department, Faculty of Science, University of Malaya,

50603 Kuala Lumpur, Malaysia

(*E-mail: rattachat.m@chula.ac.th)

Abstract The possibility of operating the 3.3 kJ United Nations University/International Centre for Theoretical

Physics Plasma Focus Facility (UNU/ICTP PFF) with a scaled down electron temperature so as to tune the

emission spectrum to the EUV region is being considered. In particular, we investigated the scaled down

operation of this plasma focus device from its original 3.3 kJ electrical input energy to as low as 960 J.

Experiments show that for discharges performed at 8 kV and with a shortened electrode length of 9 cm from

the original 16 cm, the emission was predominantly in the wavelength range of 12 to 18 nm.

Keywords plasma focus – EUV source

INTRODUCTION

The development of Extreme Ultraviolet (EUV)

radiation sources is recently gaining much interest

in semiconductor manufacturing industry due to the

expectation that the Next Generation Lithography

(NGL) will be using the wavelength of 13.5 nm [1].

Many types of EUV radiation sources, including the

laser produced plasma and pulsed discharge sources

such as the capillary discharge [2, 3], vacuum spark

[4, 5] and plasma focus [6, 7] are being considered

by researchers worldwide. These radiation sources,

especially the pulsed discharge sources are favourable

as EUV radiation source because of their lower cost

and simplicity in operation when compared to other

radiation sources.

The United Nations University/International

Centre for Theoretical Physics Plasma Focus Facility

(UNU/ICTP PFF) is a 3.3 kJ Mather type plasma

focus [8] which is optimized to produce fusion

neutrons with deuterium as the operating gas [9].

With argon or xenon as the working gas, it is known

to be capable of producing intense radiation in the

X-ray region [10-12]. The typical plasma produced

by the optimized UNU/ICTP PFF has an electron

temperature of several keV and an expected electron

density of greater than 10 19 cm -3 [8].

In this work, we consider the possibility of scaling

down the temperature of the UNU/ICTP PFF argon

plasma focus so as to operate it as an EUV radiation

source. The scaling can be done in two approaches.

The first approach is by reducing the electrical input

energy or by increasing the operating pressure while

the original geometry of the UNU/ICTP PFF is

maintained. In this case, the dynamics of the current

sheet is slowed down so that the focusing occurs

at a time after the peak of the discharge current for

electrodes with original length of 16 cm. This will

effectively make the focus less efficient although

the required electron temperature of around 110 eV

can be obtained. In the second approach, the reduced

efficiency caused by the mismatch of the dynamic

characteristic time and the electrical characteristic

time can be compensated by reducing the length of

the electrodes. A discharge voltage of as low as 6.5

kV corresponding to an electrical input energy of 634

J is shown to be sufficient.

According to the Coronal Equilibrium Model

(CEM), there are two possible ranges of temperature

at which argon plasma is expected to consist of

Jostt vol 6.indd 43 7/22/10 10:09:23 PM

43


44

Journal of Science and Technology in the Tropics (2010) 6: 43-47

ionic species that can be considered as emitters of

line radiations at wavelength around 13.5 nm. At a

temperature of around 10 to 20 eV, the Ar 6+ and Ar 7+

are prominent, whereas at a temperature of around

100 to 130 eV, the argon plasma is expected to consist

of predominantly the Ar 10+ and Ar 11+ ionic species.

The population distribution of these two groups of

ionic species as predicted by the CEM is illustrated

in Figure 1. These ionic species are known to be able

to emit intense line radiations at or near 13.5 nm as

listed in Table 1 [13].

In order to test the possibility of tuning the UNU/

ICTP PFF to produce condition which is sufficient

to produce intense EUV radiation but not X-ray, we

scaled down the operating parameters, particularly

the input electrical energy. In order to match the

slower dynamics of the current sheet due to the

lowering of the input energy, the electrode lengths of

the device were reduced to 9 cm and 7.5 cm and the

discharge voltage was reduced to 8 kV and 6.5 kV

respectively, corresponding to input energy of 960 J

and 634 J respectively. The operating pressure was

Table 1. Expected lines near 13.5 nm from argon ions Ar 6+ ,

Ar 7+ , Ar 10+ and Ar 11+ .

Ion Wavelength

(nm)

Transition Upper

level

Lower

level

Ar 6+ 13.4797 5p-3s (3s5p) 1 P 1 (3s 2 ) 1 S 0

Ar 7+ 13.5591 9f-4d (9f) 2 F 7/2 (4d) 2 D 5/2

Ar 10+ 13.57 2p-2s (2s2p 5 ) 1 P 1 (2p 4 ) 3 P 1

Ar 10+ 13.563 2p-2s (2s2p 5 ) 1 P 1 (2p 4 ) 3 P 1

Ar 11+ 13.6 2p-2s (1s 2 2p 5 ) 2 P 3/2 (2s2p 4 ) 4 P 3/2

Population

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Ar 6+

Ar 7+

Ar 10+

Ar 11+

50 100 150 200

Temperature (eV)

Figure 1. The temperature range at which argon

ions Ar 6+ , Ar 7+ , Ar 10+ and Ar 11+ are prominent

predicted by Coronal Equivalent Model.

adjusted within the range of 0.5 to 2.5 mbar for fine

tuning. Focusing action was obtained over this range

of pressure and the radiation emission in the EUV

region was observed consistently.

EXPERIMENTAL SETUP

The experimental setup is shown schematically in

Figure 2. The electrode system consisted of a central

hollow copper anode of 1.9 cm diameter, and a

cathode of six copper rods arranged in a circle of 6.4

cm diameter concentric with the anode. The anode

was insulated from the cathode at the back-wall by

a Pyrex glass tube. Three anode lengths were used,

including the original length of 16 cm, and two other

shorter lengths of 9 cm and 7.5 cm. The plasma focus

was operated with argon at pressures in the range of

0.5 to 2.5 mbar. The discharge voltage was varied

from 6 kV to 12.5 kV. The diagnostics used included

the Rogowski coil for discharge current measurement,

resistive voltage divider for transient voltage

measurement, PIN Si-diodes for X-ray measurement,

and silicon p-n junction photodiode with integrated

multilayer thin film filter for EUV measurement.

The BPX65 PIN Si-diode operated with its glass

window removed is now commonly used for the

measurement of soft X-ray from pulsed plasma due

to its fast rise-time of 2 ns [10, 12]. BPX65 diode

purchased off the shelf has a typical spectral response

of above 0.1 A/W in the wavelength range of 450 nm

to 1050 nm, with a peak spectral response of 0.55 A/W

at 900 nm [11]. However, with the front glass window

of its TO-18 casing removed, its spectral response can

be extended to the X-ray region. In order to exclude

photons in the visible region, the diode was covered

by 24 µm aluminized Mylar.

+ HV

BPX 65

SXUV

Anode

Insulator

Cathode

Figure 2. Schematics showing the experimental setup.

Jostt vol 6.indd 44 7/22/10 10:09:24 PM


A silicon p-n junction photodiode with 100 nm

silicon and 200 nm zirconium directly deposited

filter [15] was used to measure EUV radiation

in the wavelength range of 12 nm to 18 nm. The

responsivity curves of the modified BPX65 covered

with 24 µm aluminized Mylar and SXUV diode with

100 nm Si and 200 nm Zr multilayer filter, plotted on

the same graph, are shown in Figure 3. It can be seen

that the filtered BPX65 was responsive to photons

with wavelength below 1 nm, with peak response of

0.147 A/W at wavelength of 0.35 nm. On the other

hand, the silicon p-n junction diode had a clear pass

band in the range of 12 to 18 nm, with peak response

of 0.095 A/W at wavelength of 12.8 nm. While the

visible range was clearly rejected by the filter, the

response of this diode to photons in X-ray region

was not certain. In the experiments described here,

the filtered BPX65 and the filtered SXUV were used

to measure the radiation emitted from the plasma

simultaneously. They were mounted to view the

Responsivity (A/W)

Voltage (kV)

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

BPX65 with 24 µm Aluminized Mylar

SXUV filtered by 100 nm Si & 200 nm Zr

2 4 6 8 10 12 14 16 18 20

Wavelength (nm)

Figure 3. The responsivity curves of BPX65

PIN diode (with 24 µm aluminized Mylar)

plotted together with that of SXUV p-n junction

diode with 100 nm Si and 200 nm Zr.

4

2

0

Current

Voltage

-100

0.0 1.0x10 -6 2.0x10 -6 3.0x10 -6 4.0x10 -6 5.0x10 -6 6.0x10 -6 7.0x10 -6

-2

Time (s)

Figure 4. The typical current and voltage signals

of a plasma focus discharge.

200

150

100

50

0

-50

Current (kA)

plasma in the side-on location at a distance of 30 cm

from the axis of the electrode system. This allowed

us to identify without ambiguity the spectral range of

the radiation detected.

RESULTS AND DISCUSSION

In its original design, the UNU/ICTP PFF had an

electrode length L = 16 cm. For a typical focusing

discharge at discharge voltage V = 12.5 kV and at an

operating pressure P = 0.5 mbar argon, the signature

voltage spike and current dip (Fig. 4) are consistently

obtained. Under this operating condition, the current

sheet in the axial acceleration phase of the plasma

focus dynamics achieves an average speed of about

4 cm µs -1 . The X-ray emission characteristics of the

plasma produced under similar condition had been

studied and reported before [7, 9]. In this study, the

measurement of the emission was extended to the

EUV region by using a SXUV silicon p-n junction

photodiode together with a BPX65 PIN diode. The

signals obtained, together with the voltage spike of a

typical 12.5 kV discharge, operated at 0.5 mbar argon

of the UNU/ICTP PFF with an electrode length of 16

cm are shown in Figure 5

The X-ray emission for a typical focusing

discharge of the UNU/ICTP PFF at V = 12.5 kV, P

= 1.5 mbar argon and with L = 16 cm is as shown

in Figure 5. This X-ray pulse is similar to those

observed in many plasma focus devices as reported

in the literature [10-12]. It indicates the hottest phase

of the plasma produced. The occurrence of the hottest

phase of the plasma coincided with the peak of the

voltage spike. Correspondingly, the SXUV diode

registered a large EUV pulse. From the area under

SXUV (V) Voltage (kV)

30

25

20

15

10

5

0

3.0x10 -6

-5

40

30

20

10

0

-10

3.0x10 -6

-20

4.0x10 -6

4.0x10 -6

SXUV

BPX65

5.0x10 -6

5.0x10 -6

Time(s)

6.0x10 -6

6.0x10 -6

7.0x10 -6

20

10

0

7.0x10 -6

Figure 5. The voltage, EUV and X-ray signals obtained

simultaneously for a 12.5 kV discharge of a plasma focus

with electrode length L = 16 cm and operated with 1.5

mbar argon.

Jostt vol 6.indd 45 7/22/10 10:09:25 PM

BPX65 (V)

45

Journal of Science and Technology in the Tropics (2010) 6: 43-47


46

Journal of Science and Technology in the Tropics (2010) 6: 43-47

this EUV pulse, the total photon energy emitted from

the plasma (assuming isotropic source) in the range

of 12 to 18 nm could be estimated to be about 50 mJ.

While the detection of the X-ray pulse for the 12.5

kV discharge is known to indicate the formation of a

focused plasma with temperature of probably greater

than 1 keV, the absence of X-ray radiation in a 8 kV

discharge is believed to indicate a lower temperature

plasma. The set of similar signals (Fig. 5) obtained

for an 8 kV discharge at 0.5 mbar argon is shown in

Figure 6. In this discharge, the length of the electrodes

of the UNU/ICTP PFF was shortened to 9 cm. This

discharge displays several features of its radiation

emission which are distinctly different from that of

the 12.5 kV discharge. Firstly, no X-ray was detected

for this discharge indicating lower temperature

plasma. The voltage signal showed multiple spikes

with low amplitudes. The corresponding EUV signal

also consisted of multiple pulses which occurred over

duration of more than 2 µs. The peak amplitude of the

pulse, however, was much lower than those observed

for discharges at 12.5 kV. However, due to long pulse

width, the area under this pulse corresponded to total

photon energy of about 100 mJ. The input energy of

the UNU/ICTP PFF was further tuned down to 540

J by operating at condition of V = 6.5 kV and L =

7.5 cm. The output EUV pulse was insignificantly

low as compared to those operated at 8 kV discharge

voltage.

In order to fine tune the EUV output from the

plasma focus at the three discharge voltages tested,

namely V = 12.5 kV, 8 kV and 6 kV, the operating

SXUV (V) Voltage (kV)

5

4

3

2

1

0

-1

3.0x10 -6

5

4

3

2

1

0

-1

3.0x10 -6

4.0x10 -6

4.0x10 -6

SXUV

BPX65

5.0x10 -6

5.0x10 -6

Time(s)

6.0x10 -6

6.0x10 -6

7.0x10 -6

5

4

3

2

1

0

-1

7.0x10 -6

Figure 6. The voltage, EUV and X-ray signals

obtained simultaneously for a 8 kV discharge of a

plasma focus with electrode length L = 9 cm and

operated with 0.5 mbar argon.

BPX65 (V)

pressure was varied from 0.5 mbar to 2.5 mbar for

a series of discharges. From the area under the EUV

pulse, the total photon energy emitted in the range of

12 to 18 nm was estimated and plotted (Fig. 7). It can

be seen that discharges with higher input energy of

2.3 kJ was able to produce EUV pulses with higher

amplitudes and shorter duration, but the total energy

corresponding to the photons emitted within the

spectral range of 12 to 18 nm was lower than those

emitted from discharges at 960 J input energy. EUV

energy of up to 100 mJ can be obtained from a 8 kV

discharge operated with 0.5 mbar argon.

CONCLUSION

By adjusting the operating parameters of the UNU/

ICTP PFF operated with argon from its originally

designed values of discharge voltage V = 15 kV,

electrode length L = 16 cm and P = 0.5 to 2.5 mbar

argon, we have obtained the possible experimental

conditions to operate it to produce plasmas with

conditions sufficient for emission up to the EUV

spectral range of 12 to 18 nm. The radiation energy

corresponding to this spectral range emitted from

a discharge with V = 12.5 kV, L = 16 cm and P =

0.5 to 2.5 mbar may be up to 95 mJ. This emission,

however, is accompanied by emission of X-ray

photons in the wavelength range of 0.3 to 3 nm. The

plasma produced under this condition is expected

to have achieved an electron temperature of up to

several keV. However, when the same plasma focus

device is operated at a scaled down condition of V

Figure 7. The variation of output EUV energy for

three sets of operation condition of discharge voltage

V and electrode length L at various pressure P. (i) V

= 12.5 kV, L = 16 cm; (ii) V = 8 kV, L = 9 cm; and (iii)

V = 6.5 kV, L = 7.5 cm.

Jostt vol 6.indd 46 7/22/10 10:09:27 PM


= 8 kV, L = 9 cm and with operating pressure in the

range of P =0.5 to 2.5 mbar argon, up to 102 mJ of

EUV photon energy can be produced and in this case,

no X-ray photon is produced. We expect the electron

temperature achieved in this case to be in the range of

several tens electron-volts. If one is currently having

the UNU/ICTP PFF and intend to use it as an EUV

source to test for the effect of EUV on a substract

which has good absorption in both the X-ray and

EUV regions, it may be necessary to eliminate the

X-ray component of the emission. In this case the

1. Banine V. and R. Moors (2004) J. Phys. D: Appl. Phys.

37: 3207.

2.

3.

Mohanty S.R., Robert E., Dussart R., Viladrosa R.,

Pouvesle J.M., Fleurrier C. and Cachoncinlle C.

(2003) Microelectron. Eng. 65: 47.

Hong D., Dussart R., Cachoncinlle C., Ressenfeld

W.E.S., Gotze S., J. Pons J., Viladrosa R., Fleurier C.

and Pouvesle J.M. (2000) Rev. Sci. Instrum. 71: 15.

4. Xiaoming G., Meisheng X., Rubin Y., Chaofeng H.,

Wirpszo K.W. and Emilio P. (2001) Proc. SPIE 4343:

491.

5. Chew S.H. and Wong C.S. (2006) J. Sci. & Technol. in

the Tropics 2: 125.

6.

7.

Fomenkov I.V., Ness R.M., Oliver I.R., Melnychuk

S.T., Khodykin O.V., Bowering N.R., Rettig C.L. and

Hoffman J.R. (2003) Proc. SPIE 5037: 807.

Rawat R.S., Zhang T., Phua C.B.L., Then J.X.Y.,

Chandra K.A., Lin X., Patran A. and Lee P. (2004)

Plasma Sources Sci. Technol. 13: 569.

REFERENCES

scaled down operation of the existing UNU/ICTP

PFF may be a possible solution. With this possibility

demonstrated, it is still necessary to point out that the

device can be further tuned to obtain the condition for

optimum EUV output.

Acknowledgements – The authors would like to extend

their great appreciation to Asian African Association for

Plasma Training (AAAPT) for its support to initiate plasma

focus research in this laboratory and to the Graduate

School, Chulalongkorn University for financial support.

8. Lee S., Tou T.Y., Moo S.P., Eissa M.A., Gholap A.V.,

Kwek K.H., Mulyodrono S., Smith A.J., Suryadi S.,

Usada W. and Zakaullah M. (1988) Am. J. Phys. 56:

62.

9. Yap S.L., Wong C.S., Choi P., Dumitrescu C. and Moo

S.P. (2005) Jpn. J. Appl. Phys. 44: 8125.

10. Favre M., Lee S., Moo S.P. and Wong C.S. (1992)

Plasma Sources Sci. Technol. 1: 122.

11.

Mohammadi M.A., Verma R., Sobhanian S., Wong

C.S., Lee S., Springham S.V., Tan T.L., Lee P. and

Rawat R.S. (2007) Plasma Sources Sci Technol. 16:

785.

12. Ng C.M., Moo S.P. and Wong C.S. (1998) IEEE Trans.

Plasma. Sci. 26: 1146.

13. http://Spectr-w3.snz.ru

14.

http://129.105.69.13/datasheets/Optoelectronics/

Photodiode_UDT_catalog.pdf

http://www.ird-inc.com

Jostt vol 6.indd 47 7/22/10 10:09:28 PM

15.

47

Journal of Science and Technology in the Tropics (2010) 6: 43-47


Journal of Science and Technology in the Tropics (2009) 5: 133-139

Jostt vol 6.indd 48 7/22/10 10:09:28 PM


Journal of Science and Technology in the Tropics (2010) 6: 49-52

An atmospheric pressure non-thermal plasma jet in nitrogen for

surface modification of polyethylene

D. P. Subedi1 , R. B. Tyata1 , A. Shrestha1 , D. Baral1 , D. K. Madhup1 and C. S. Wong2 1Department of natural Sciences, School of Science, Kathmandu University, Nepal

2 Plasma Research Laboratory, Department of Physics, University of Malaya, 50603 Kuala Lumpur,

Malaysia

Abstract In this paper, an atmospheric pressure plasma jet (APPJ) in nitrogen has been set up for possible

application in polymer surface modification. The effect on the hydrophilicity of a polyethylene (PE) surface

exposed to the plasma jet was investigated for different exposition times and distances from the nozzle. It has

been confirmed that the jet can modify polymer film with a work distance of more than 60 mm.

Keywords non-thermal plasma jet – surface modification – polyethylene – polymer film – hydrophilicity

INTRODUCTION

In the last one decade, research on non-thermal

plasma at atmospheric pressure has become a subject

of great interest because of its applications in various

fields. The characteristic of these types of plasma is

the existence of thermal non-equilibrium between the

electrons and the ions. The treatment of materials

by non-thermal atmospheric pressure plasma is a

promising technology that is simple to set-up, easy

and economical to operate and does not require

vacuum equipments.

Among the various applications of the nonthermal

atmospheric pressure cold plasmas are

sterilizations of living tissue without damage and

blood coagulation [1], modulation of cell attachment

[2], biological and chemical decontamination [3-5],

water decontamination and pollution control [6,7],

nanotechnology [8], surface cleaning, etching, thin

film deposition, surface modification and material

processing [9-13]. Atmospheric pressure plasma can

be generated by various methods: corona, glow, arc,

dielectric barrier discharge (DBD), RF discharge

and microwave discharges. The main problem with

these systems is that the working space is often

limited because of the electrode configuration. In

an atmospheric pressure plasma jet, the plasma

constituents are expelled through an orifice by a gas

flow which makes it possible for the treatment of large

objects. Forster et al. [14] reported an atmospheric

pressure plasma jet in a DBD configuration. This type

of plasma can be operated under high gas flow rate.

In 2003, Toshifuji et al. [15] reported a relatively cold

arc plasma jet produced under atmospheric pressure

having potential application for surface modification.

Quite recently, Takemura et al. [16] developed an

atmospheric pressure plasma jet with long flame which

can modify polymer film with a work distance of

over 200 mm. Recently, a double layered atmospheric

pressure plasma jet had also been reported [17].

In the present study, a non-thermal nitrogen

plasma jet was generated using a high voltage

power supply with output frequency of 10 to 30 kHz

under atmospheric pressure. The application of this

plasma jet to treat surface of polymer material is also

demonstrated.

EXPERIMENTAL SETUP

The schematic diagram of the plasma jet system

is depicted in Figure 1. A brass rod of diameter 3

mm was placed inside a glass tube of inner diameter

4 mm and outer diameter 6 mm. At one end of the

glass tube a steel cap electrode (9 mm diameter, 0.5

mm thickness and 20 mm long) with an orifice of

2 mm diameter, was mounted. The inner electrode

was connected to high voltage (0-20 kV) and high

frequency (10-30 kHz) power supply and the outer

electrode was grounded.

In this study, nitrogen is used as the working gas.

Jostt vol 6.indd 49 7/22/10 10:09:28 PM

49

Journal of Science and Technology in the Tropics (2009) 5: 133-139


50

Journal of Science and Technology in the Tropics (2010) 6: 49-52


Digital
Oscilloscope


N2


In order to evaluate the effectiveness of the plasma

jet in modifying the PE surface, hydrophilicity test

was performed. The contact angle of water droplets

at the surface of the PE film was measured using a

rame’-hart Contact Angle Goniometer. This unit

was equipped with standard software to analyze the

drop image for the calculation of surface energy.

The system offered a high level of computer aided

precision in measuring contact angle and therewith

facilitating the calculation of surface energy using

different model equation.

The water droplet contact angles on the surface

of PE films treated at different distances from the

nozzle of the jet and for different exposure times were

measured. Each contact angle presented in this paper

was an average of at least four measurements made

on different positions on the surface of PE.

RESULTS AND DISCUSSION

PC.
DAQ.


Voltage
Probe


Current
Probe


An example of the plasma jet obtained is shown in

Figure 2. A discharge between the centre electrode

and the steel cap containing the orifice is expected to

be produced and the jet is formed due to the flowing

nitrogen stream. The electron temperature of the

plasma near to the orifice is expected to be several

electron-volts. However, the temperature of the ions

and the neutral atoms/molecules is relatively cold and

was measured by using an IR thermometer to be in

the range of 28°C to 30°C. The length of the jet was

found to be dependent on the gas flow rate (Fig. 3).

The flow rate of nitrogen was varied from 1 to 5

litres per minute. To measure the length of the jet,

a scale was placed behind the jet while taking the

Arc
Plasma
Jet


Emission


Spectrometer


Figure 1. Experimental setup of the plasma jet.

Figure
1.
Experimental
setup
of
the
plasma
jet.


High
Frequency


Power
Supply


image of the jet by a digital camera. The lengths were

then determined from the photograph. It is clear that

the length of the jet increases proportionally with the

gas flow rate at the beginning and then levels off after

4 litres per minute (Fig. 3).

Length of visible jet [mm]

Figure 2. A picture of the non-thermal plasma jet.

75

70

65

60

55

1 2 3 4 5

Gas flow rate [l/min]

Figure 3. Dependence of the jet length of APPJ on

the flow rate of nitrogen.

Jostt vol 6.indd 50 7/22/10 10:09:30 PM



Contact angle [Deg.]

90

80

70

60

50

40

0 20 40 60 80 100

Distance from the orifice [mm]

Figure 4. Water contact angle of the PE film as

a function of distance from the orifice of APPJ.

The gas flow rate was 3.0 l min -1 and the exposure

time was 10 s.

Figure 4 shows the water contact angle of the

PE film as a function of distance from the orifice

of APPJ. In order to investigate the effectiveness of

PE surface modification by the APPJ, hydrophilicity

tests were carried out on the PE films before and after

the treatment. At least three drops of the test liquids

were placed on the sample and the contact angles

were measured from the profile of the drops. The

untreated sample had contact angle of 98º with water.

Whereas after the exposure to the jet, a minimum

contact angle of 39º was measured for sample placed

at a distance of 5 mm from the orifice for a time of

10 s. For the same exposure time, a contact angle of

82º was measured when the sample was placed at a

distance of 100 mm from the orifice.

Figure 5 shows the contact angle of PE sample

as a function of the exposure time in APPJ. The

1. Fridman G. (2006) Plasma Chemistry and Plasma

processes 26: 425.

2. Stoffels, E., Kieft I. and Sladek R. (2003) J. Phys. D:

Appl. Phys. 36: 2908.

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8.

Kolb J.F., Joshi R.P., Xiao S. and Schoenbach K.H.

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Gonzales-Aguilar J., Moreno M. and Fulcheri L.

Contact angle [deg.]

REFERENCES

100

90

80

70

60

50

40

treatment time ranged from 1 s to 12 s. As shown in

the figure, the contact angle of the film changed from

98º for the untreated to the lowest value of 43º after

the treatment.

CONCLUSION

0 2 4 6 8 10 12

Exposure time [s]

Figure 5. Water contact angle of PE sample as

a function of the exposure time in APPJ. The

distance of the sample from the orifice was 5 mm

and the gas flow rate was 3.0 l/min.

An atmospheric pressure plasma jet has been

developed and tested. The irradiation of the jet onto

the polymer surface can improve the hydrophilicity

of the sample without any damage to the material.

The advantages of the present jet system are that the

power consumption is low and there is no need of

matching network. This plasma source can be used

for the treatment of non-planar surface and heat

sensitive materials.

(2007) J. Phys. D: Appl. Phys. 40: 2361.

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10. Kunhardt E.E. (2000) IEEE Trans. Plasma Sci. 28:

189.

11. Daun Y., Hung C. and Yu Q.S. (2005) IEEE Trans.

Plasma Sci. 33: 328.

12. Borcia G., Chiper A. and Rusu I. (2006) Plasma

Sources Sci. Technol. 15: 849.

13. Benedict J., Forcke K., Yanguas-Gil A. and Keudell

von A. (2006) J. Appl. Phys. 99: 112.

14. Foster S., Mohr C. and Viol W. (2005) Surface and

Coatings Technology 200: 827.

15. Tioshifuji J., Katsumata T., Takikawa H., Sakakibara

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Journal of Science and Technology in the Tropics (2010) 6: 49-52


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Journal of Science and Technology in the Tropics (2009) 6: 49-52

16.

17.

T. and Shimizu I. (2003) Surface and Coating

Tech. 171: 302.

Takemura Y., Kubota Y., Yamaguchi N. and Hara T.

(2009) IEEE Trans. Plasma Sci. 26: 1604.

Choi J., Matsuo K., Yoshida H., Namihira T., Katsuki

18.

S. and Akiyama H. (2009) Japanese J. Appl. Phys. 48:

086.

Subedi D.P., Madhup D.K., Adhikari K. and Joshi

U.M. (2008) Indian Journal of Pure and Applied

Physics 6: 540.

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Journal of Science and Technology in the Tropics (2010) 6: 53-57

Compressed natural gas (CNG) cylinder testing and data evaluation

using acoustic emission technique

Tonphong Kaewkongka 1,* , Jirapong Lim 2 and Suparerk Sirivedin 3

1Department of Physics, Faculty of Science, Chulalongkorn University, Patumwan,

Bangkok 10330, Thailand

2Department of Production Engineering, Faculty of Engineering, King Mongkut University of Technology,

North Bangkok, Bangsue, Bangkok 10800, Thailand

3King Mongkut University of Technology, North Bangkok, Bangsue, Bangkok 10800, Thailand

(*E-mail: tonphong.k@chula.ac.th)

Abstract This paper describes a method of compressed natural gas (CNG) storage cylinder testing using

acoustic emission technique. The CNG storage cylinder, which is type I-steel cylinder, was tested as

normal and predefined surface crack operating condition. Acoustic emission (AE) signals propagation and

transmissions were captured by piezoelectric transducers mounted on surface across the cylinder with and

without predefined surface crack. In the experiment, an increased step-wise condition in hydrostatic pressure

up to 400 bars was applied to the cylinder. The feature extraction and classification of AE signals from each

testing condition was applied. The results are very promising in terms of identifying the defects due to crack

propagation.

Keywords acoustic emission – CNG cylinder

INTRODUCTION

The number of vehicles using compressed natural gas

(CNG) has grown rapidly for the past decades due to

the alternate use of energy and become an important

issue in Thailand. In most cases, the nature of the

contents renders these cylinders critical to operations

and safety. There have been several severe accidents

from the explosion of CNG cylinders. Therefore, it

may lead to significant loss or catastrophic failures.

For health and safety issue, monitoring the condition

of the cylinders has received considerable attention

over the past few years due to its particular importance

of safety, environment and the economical reason [1-

3]. A reliable condition monitoring system will reduce

the failure and possible loss.

According to inspection standard, it requires that

cylinders be visually inspected externally after it

has been used for 36 months. This is to ensure that

the cylinders that have damage or deterioration will

be removed from service or repaired. The visual

inspection test needs removal of the cylinder from

the vehicle. However, there are possible risks if the

internal flaws or defects cannot be examined.

Nowadays there are many kinds of conventional

methods available for the CNG storage cylinder

detection [4-6]: visual inspection using dye penetration,

hydro-static testing, flow or pressure measurement,

ultrasonic testing. However, these methods are time

consuming due to the requirement of removing the

cylinder from the vehicle and revealing the defects of

the cylinder after it had already occurred. Recently,

acoustic emission (AE) has been extensively applied

to vessels testing during pressurization and has proven

to be a timely and economical method for structural

integrity assessment as described in the standard and

code, ISO/DIS 16148.2 [7]. A major advantage of AE

inspection is that it allows the whole structural integrity

to be tested non-intrusively with minor disruption (no

need to remove the cylinder from service, empty and

clean for inspection) in a pressurized condition. AE

can provide complete coverage of the cylinder with

pressurized test and is sensitive to active defects or

flaws presence and propagation.

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Journal of Science and Technology in the Tropics (2009) 5: 141-150


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Journal of Science and Technology in the Tropics (2010) 6: 53-57

The objective of this research is to demonstrate that

a condition-based monitoring using acoustic emission

(AE) can provide not only timely detection of defects

in the cylinder but also the crack propagation so that

maintenance or replacement can be performed prior

to the loss of safety function. Therefore the use of

acoustic emission method has been proposed for

CNG type-I steel cylinder monitoring instead of the

conventional methods.

EXPERIMENTAL SETUP AND APPARATUS

Acoustic emission is a natural phenomenon of stress

wave generation and propagation spontaneously

when a material is subjected under stress [8]. Plastic

deformation and growth cracks are the primary

sources of acoustic emission in metals. The acoustic

signal can be detected by a piezoelectric transducer,

which converts the mechanical energy carried by the

elastic wave into an electrical signal.

When the defective cylinders are pressurized,

stress waves (AE) can be produced by several different

sources (e.g. secondary sources or actual propagation

of cracks). These sources can result in AE activities

generated at pressure less than, equal to or greater

than the operating pressure. The stress waves are then

propagated throughout the structure.

A systematic approach to classify the dynamic

responses of AE signatures associated with the

CNG type I steel cylinder operating condition was

performed in this study (Fig. 1). The AE testing is

usually carried out during a controlled pressurization

of a cylinder. Conventional AE parameter, AE rms is

used to identify the presence of acoustic emission

activities produced when microscopic cracks occur.

The AE rms can be calculated using the formula below

[9]:

where v ( t)

is the electrical voltage signal obtained

from piezoelectric transducer, t o is the initial time,

T is the period of the AE activity.

Figure 1. A system of acoustic emission testing.

Figure 2. Set up of CNG cylinder testing with four installed AE

sensors.

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The acoustic emission sensors, model AE-

SS1 (Holroyd Instruments, UK) which were made

of piezoceramic elements, were mounted on the

circumference along the surface of the cylinder

(Fig. 2). The acoustic emission sensors of 100 kHz

resonance frequency responded well with the material

degradation and microscopic crack initiation and

growth. The captured acoustic emission signals were

then conditioned and amplified with 60 dB-gain signal

conditioning unit. The PCI-1714UL was used for

A/D conversion and data logger. In the experiments,

a normal operating cylinder was tested with four AE

sensors by means of mechanical coupling (Fig. 2).

The cylinder used in this experiment had a 60 litre

capacity. It was made of a steel sheet with a diameter

of 300 mm, length of 990 mm and thickness of 8

mm.

To perform hydrostatic test, the cylinder was

placed in a chamber which could facilitate a high

pressure testing (Fig. 3). For an abnormal operating

condition cylinder, a simulated surface crack was

performed on a good cylinder. The simulated

(artificial) crack (Fig. 4) was made using a grinder to

generate a surface defect with length of 30 mm (along

the length of cylinder), width of 3 mm and depth of

2 mm.

To eliminate background noise, the threshold of

AE signal was set to 39 dB AE . Evidently, the threshold

level affects the value of the AE parameters. A typical

Figure 3. Installation of CNG cylinder in a

high-pressure hydrostatic test chamber.


example is the event duration. By definition, it is the

time that the AE event is above the threshold. A step

wise increase in hydrostatic pressure was performed

up to 400 bars with the testing cylinder. During the test,

a pressure sensor was attached with the hydro-testing

machine as for load reference for data acquisition.

EXPERIMENTAL RESULTS AND

DISCUSSION

To verify the proposed approach, the experiment on

the normal operating cylinder was performed using

hydrostatic test. A hydrostatic test was applied on the

testing cylinder with increased pressure up to 300

bars. The AE signals captured from all four sensors

Figure 4. Artificial surface crack generated

at the side wall of the cylinder.

Figure 5. AE signals versus time (data point)

from a normal CNG cylinder.

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Journal of Science and Technology in the Tropics (2010) 6: 53-57


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Journal of Science and Technology in the Tropics (2010) 6: 53-57

attached on the good or normal operating condition

cylinder yielded a calm condition in a random

background noise fashion (Fig. 5).

In the experiment, an artificial surface crack

(300mm length, 2 mm width) cylinder was tested with

the applied pressure up to 300 bars. The experimental

results are shown in Figure 6. It illustrates that the

time domain signals of the artificial defective wall

cylinder gave rise to the abrupt change in higher

amplitude to all four AE sensors.

Ultimately, the pressure was increased up to

400 bars which resulted in the crack of the artificial

defect of the wall. At this state, the water was leaking

out due to the open surface of the previous artificial

crack because it could not sustain the increased the

internally applied load any further. This resulted in

the AE signals as shown in Figure 7.

The AE rms values calculated from the time domain

signal are shown in Figure 8. For the normal cylinder,

it gave the lowest signal energy around 15.8 mV.

This is because there was only background level of

the hydrostatic test that was present. It illustrates

that the defective cylinder condition yielded the

highest value about 211 mV. Its value was about 13.3

times greater than the one from normal operating

condition. This is due to the microscopic crack growth

as the primary source and the turbulent flow at the

surface of the crack as secondary source. Whereas

the leakage cylinder shows that the AE rms values were

relatively greater than the normal condition at about

20.8 mV (greater by 5 mV).

The methodology described in this paper has

been shown to work well for monitoring CNG

cylinder operating conditions. The method involves

calculation of AE rms values as a mean to identify the

abnormal operating conditions of the CNG cylinder

together with hydrostatic test. Overall the proposed

method can provide timely detection of the presence

of crack and leakage of the CNG cylinder and is

efficient to implement.

1. ASME (2000) Boiler and Pressure Vessel Section XI,

Appendix A, Article A-3000.

2. ASTM E2191-08 (2006) Standard Practice for

Examination of Gas-Filled Filament – Wound

Composite [3] Pressure Vessels Using Acoustic

Emission.

REFERENCES

Figure 6. AE signals versus time (data point) from a

defective CNG cylinder.

Figure 7. AE signals versus time (data point) from a

leaked CNG cylinder.

Figure 8. Results of AE rms from different operating

conditions.

3. ASTM E1419-02b (2002) Standard Test Method for

Examination of Seamless, Gas-Filled, Pressure Vessels

Using Acoustic Emission.

4. Connolly M.P. and Han Dinh (1996) Fleet Inspection

of Compressed Natural Gas Cylinders for Natural Gas

Vehicles Using Source Location Acoustic Monitoring.

Jostt vol 6.indd 56 7/22/10 10:09:50 PM


SAE Technical Paper Series No. 961174.

5. Webster C. (2007) Development of Non-Destructive

Evaluation (NDE) Techniques for CNG Fuel Tanks.

Report for Transportation Development Centre of

Transport Canada.

6. Hudak S.J., Jr. (1991) Assuring the Safety of Natural

Gas Vehicles. Technology Today Magazine, Sept.

1991.

7. ISO/DIS 16148.2 (2006) Gas cylinders – Refillable

seamless steel gas cylinder – Acoustic emission

examination for periodic inspection.

8. Pollock A.A. (1973) Acoustic Emission 2 – Acoustic

emission amplitudes. Journal of Non-Destructive

Testing10: 264-269.

9. Scruby C.B. (1987) An introduction to acoustic

emission. Journal of Physics Instrument Science and

Technology 20: 946-953.

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Journal of Science and Technology in the Tropics (2010) 6: 53-57


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Journal of Science and Technology in the Tropics (2010) 6: 59-65

Prompt gamma neutron activation analysis (PGNAA) of hydrocarbons:

A Monte Carlo study with GEANT4

Lam YiHua1 , Wong Chiow San1 * and Kurunathan Ratnavelu2 1Plasma Research Laboratory, Department of Physics, Faculty of Science, University of Malaya,

50603 Kuala Lumpur, Malaysia

2Institute of Mathematical Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

*Corresponding author

Abstract In the present work, the results of prompt gamma (γ) neutron activation analysis (PGNAA)

calculated by the open source GEANT4 Monte Carlo toolkit were compared with the commercial simulation

package MCNP. Both the Monte Carlo simulation packages utilize the phenomenological nuclear reactions

to yield γ rays which are coupled with incident neutrons after these reactions. A virtual experimental setup

with a 4π solid-angle γ detection was instantiated in the simulations to collect all prompt γ rays from the

tested hydrocarbon phantoms. The comparative results present good agreement in getting the characteristic

γ energy spectra of hydrocarbon materials.

Keywords GEANT4 – MCNP – Monte Carlo simulations – prompt gamma neutron activation

INTRODUCTION

A material with its specific chemical composition and

density can be detected or differentiated from among

other types of materials, which have nearly the same

chemical composition but have not complied with the

same densities, by using prompt gamma (γ) neutron

activation analysis (PGNAA) method. It has been

used in detecting explosive hydrocarbon material [1].

In addition, certain element in a bulk material or in a

biological tissue can be determined via PGNAA, e.g.

biomedical protein detection [2].

PGNAA method relies on neutron-nuclear

interactions, that is, inelastic scattering (n, n′γ), (n, pγ)

and radiative capture (n,γ), to yield γ rays. Although

1/v law [3] (v is the incident neutron velocity) is not

applicable to every element, isotope and energy range

[4], it is still acceptable to be applied in low Z element

and in thermal energy range [5]. This law infers that

the probability of neutron radiative capture depends

on how long the period of time a neutron spends in

the region surrounding the nucleus. The longer period

of time it spends, the higher probability of neutron

radiative capture will occur. Hence, when a low

kinetic energy incident neutron, e.g. thermal neutron

(~0.025eV) or epithermal neutron (0.1-1eV), enters the

narrow separation virtual energy levels in compound

nucleus or interacts with compound nucleus [6], the

neutron radiative capture cross sections will increase.

These low energy interactions raise the possibility of

producing stable isotopes and the coupled γ ray, e.g.

1 H(n, γ) 2 H in which 2 H is a stable isotope and γ ray will

be radiated. Besides, different materials composed by

different chemical compositions (including different

isotopes) and densities will have different radiative

capture cross sections. By radiating low energy

neutrons with certain range of energy distribution on

different materials, the produced γ ray energy spectra

will be different. Hence, the prompt γ ray energy

spectra become an identifying characteristic of the

material.

Other than experimental measurements, PGNAA

can be simulated by using various types of Monte

Carlo computational simulation packages, such as

GEANT4 [7,8], MCNP [9] and FLUKA [10,11].

Most of these packages are written in structural

programming language (C and FORTRAN). GEANT

4 has been written in object-oriented programming

(OOP) architecture (C++), developed and maintained

by many laboratories [7,8]. Conceptually, OOP has

its distinctive feature in treating particle as a single

object which may have encapsulated its own data

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Journal of Science and Technology in the Tropics (2010) 6: 59-65

structure (characteristic data and methods) and

respective particle transport physics model. The OOP

methodology (the way in composing program) can

resemble the physical occurrence, e.g. a neutron,

which is treated as an object in the simulation,

may collide and be captured by a thermal nucleus

(the second object) to yield γ ray (the third object);

alternatively, the neutron may undergo a beta decay

process to produce a proton (the second object), an

electron (the third object) and an anti-neutrino (the

fourth object). The created electron may produce

γ ray (the fifth object) during Bremsstrahlung

process. During radiative decay, annihilation and

radiative capture, the interacting particles will be

deleted from the computer memory after yielding

respective secondary particle(s). In this way, the OOP

computational method resembles the physics process

as the active interacting particle, e.g. electron, positron

and neutron, will disappear after its respective physics

process, e.g. annihilation or radiative capture.

In this work, the incident neutrons are assumed

to be thermalized and their energies are in accord

with Maxwell-Boltzmann (MB) distribution, and the

incident energy of highest probability is 0.025 eV. The

prompt γ energy spectra of rubber, which are calculated

by GEANT4 by means of either inducing thermalized

incident neutrons or inducing monoenergetic incident

neutrons on target, is compared with the results of

prompt γ energy spectra induced by monoenergetic

incident neutrons, which was generated by the MCNP

[12].

SIMULATION OF PGNAA

Physics list and incident random energy

generator

The calculations of the current PGNAA Monte Carlo

simulation were processed by GEANT4.8.2.p01,

Detector


Construction


Virtual


Experimenta

l
Setup


Registere

Registered

d


Materials Materials


G4NDL3.10,
etc


GEANT4


Graphical


Visualization


General
Particle
Source



(Maxwell­Boltzman
Distribution)


CLHEP
random
number
generator


Figure 1. Schematic organization of PGNAA simulation program.

whereas the evaluated neutron cross section input

data was by G4NDL3.10, a recompiled data of a few

evaluated nuclear data (including ENDF-B VI, JENDL,

FENDL, CENDL, Brond, EFF, JEF, MENDL) [7].

Besides, data sets such as G4EMLOW2.3 were used as

part of the input data to facilitate the calculation of the

γ propagation handled by electromagnetic processes

(Low Energy Electromagnetic). These data files were

input via respective physics process classes. Figure 1

shows the schematic flow of the application program

of GEANT4.

Neutron Elastic, Inelastic, Capture and Fission

handled by the neutron high precision (HP) model

[7], [13] are considered in this PGNAA simulation.

These four low energy neutron physics models are

able to describe high precision final state production

which is based on the G4NDL3.10. It covers the

neutron scattering energy range from thermal energy

up to 20 MeV. In addition, Doppler broadening and

thermal motions of target nucleus are covered in the

final state generator. Both processes are done on-thefly

by the neutron HP models and their associated

cross sections, G4NDL. Although fission is quite

rare to happen, it is included in the simulation as a

precaution in the PGNAA simulation. These four

processes are considered as post-step reactions and

one of them is selected via random number selection

by means of comparing the mean free path. Before

the selection of physics processes, an element will

be selected among elements composing a material

and an isotope will be selected from among isotopes

which are defined for that element. These two levels

of selection (of element and isotope) are calculated

via random number selection and comparison of

the respective cross sections in GEANT4 kernel

(class G4HadronicProcess). However, inside the

class G4HadronicProcess, the original method

G4HadronicProcess::ChooseAandZ assumes isotope

Stepping


Action


ASCII


Output
Results


(γ
energy)


Analysis


Manager


Output
Results



(γ
energy)


AIDA
Interface



Grace5.1.21


(Data
Analysis)


JAS3


(Data
Analysis)


Jostt vol 6.indd 60 7/22/10 10:09:53 PM


abundance values by retrieving the default values in an

instantiated object of G4StableIsotope. These isotope

abundance values may not suit the user need. Hence,

the method G4HadronicProcess:ChooseAandZ

had been altered to suit the user defined isotope

abundance values [14].

Besides, the simulations have been separately run

with two sets of electromagnetic physics models: (1)

Standard Electromagnetic physics model; and (2)

Low Energy Electromagnetic physics models. Both

electromagnetic physics models consist of e - e + pair

production, photoelectric, γ conversion and Compton

scattering. Extra physics models have been attached

to e - and e + : (1) Multiple Scattering; (2) Low Energy

Ionization and Low Energy Bremsstrahlung; or

Standard Ionization and Standard Bremsstrahlung;

whereas Low Energy Rayleigh has been attached

to γ. However, we find that both sets of physics

model – Standard Electromagnetic and Low Energy

Electromagnetic produce the same outcomes.

Therefore, only one set of results is shown in this

paper. Furthermore, the registered processes for

proton and ions (deuteron, triton, 3 He, alpha, generic

ion) are Multiple Scattering, Low Energy Ionization

and respective hadronic routines e.g. low energy

proton/deuteron/triton/alpha inelastic and elastic

processes.

We assume the process of neutron thermalization

will eventually produce neutrons with energy

distribution described by Maxwell-Boltzman (MB)

distribution,

(1)

Instead of integrating the distribution function to

yield the randomized incident neutron energies, the

function can be treated in the form of a histogram

with 10000 or more bins. Then the histogram can

be normalized to its maximum value. The output

of this function f(E) can be cumulated as a discrete

cumulative distribution [15]. Even random numbers

between 0 and 1, which are generated via a Class

Library for High Energy Physics (CLHEP-2.0.3.1),

are distributed on the ∑f(E)-axis. The corresponding

x-axis values are the MB randomized incident

neutron energies. The MB distribution code has

been embedded into a new class inherited from

the class G4GeneralParticleSource. The highest


probability energy of the random distribution is

0.025eV within the energy range of 0.001eV < E <

0.050eV.

Virtual experimental setup

The listed isotopes in Table 1 and materials in

Table 2 were constructed in a user defined detector

construction class and registered into the GEANT4

kernel. Figure 2 shows the experimental setup, which

consisted of two spherical volumes (phantoms), i.e.

the internally occluded sphere (black, with radius

0.62 cm) and the exterior occluding hollow sphere

(grey, with radius 1.05 cm). The setup was similar

to Nunes et al. [12] and Sohrabpour et al. [16]. The

hollow sphere and the inner spheres could be either

set as the same material or set as different materials.

The total physical volume of both spheres was 1

cm 3 . Therrmalized neutrons would be directed in

Table 1. List of registered isotopes.

Registered isotopes Natural abundance (%)

1 H 99.985

2 H 0.015

12 C 98.80

14 C 1.20

16 O 99.757

17 O 0.038

18 O 0.205

14 N 99.640

15 N 0.360

Table 2. List of registered materials.

Registered Chemical Density

materials composition (g/cm3 Temperature

) (K)

C4

Rubber

C H O N 4 6 6 6

C H 5 8

1.83

0.94

273.15

273.15

Figure 2. A typical run sample (snapshot) with

prompt γ rays.

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Journal of Science and Technology in the Tropics (2010) 6: 59-65


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Journal of Science and Technology in the Tropics (2010) 6: 59-65

parallel to the both spheres. The source of neutrons

was confined in a plane square (2.5 × 2.5) cm 2 , and

located 5 cm (arbitrary) from the spheres. As long

as the yield γ ray of inelastic (n, n'γ) or capture

(n,γ) reaction propagated from spherical volumes to

atmosphere in 4π directions, energy of the prompt γ

ray would be cumulated for further analysis. There

was no variance reduction applied on the analysis.

During the simulation, random neutron incident

energy and energy of prompt γ ray results were kept

in ASCII and Abstract Interfaces for Data Analysis

(AIDA) 9170 format files for further analysis which

was respectively based on Grace and Java Analysis

Studio (JAS3).

Figure 2 shows a typical run of the simulation.

Both spherical volumes (inner and outer) were set as

explosive C4, and they were placed in the ambience of

atmospheric gas (70% N 2 , 30 % O 2 ), alternatively, the

ambience could be set as vacuum too. The spherical

volumes were exposed to 100 incident neutrons with

MB energy distribution. Light grey lines represent

the neutron tracks (incident neutrons or scattered

neutrons). This typical run produced two γ tracks

(grey lines) as labeled in Figure 2. The chemical

composition of C4 is given in Table 2.

RESULTS AND DISCUSSION

In this PGNAA simulation, γ energy spectra of

a typical plastic explosive (C4), rubber, and C4

occluded by rubber were calculated and compared.

Figures 3-5 show the comparison of γ energy spectra

produced by GEANT4.8.2.p01 and MCNP (analyzed

by Nunes et al.) [12]. The γ energy spectra represent

the interactions between the thermal neutrons and

the nucleus of the constituent elements of C4 and

rubber, and represent also γ interactions in the sample

material itself. The ratio of non-neutron-capture

(i.e. ionization, Bremsstrahlung, transportation,

annihilation, radioactive decay and multiplescattering)

processes, which are related to γ yield, to

neutron capture process is ~ 0.08. It may be infered

that thermal neutron capture process plays a significant

role in γ production. The identity of the constituents

of the particular material can then be adduced. There

are three configurations of virtual experimental

setup: (1) both inner and outer spherical phantoms are

placed in atmospheric environment and are irradiated

by MB distributed energy neutrons (the upper most

histogram); (2) both spherical phantoms are located

in vacuum and are irradiated by MB distributed

Figure 3. Prompt γ energy spectra of C4 generated by GEANT4.8.2.p01 compared with MCNP.

Jostt vol 6.indd 62 7/22/10 10:10:09 PM


energy neutrons (the second upper histogram); and

(3) both spherical phantoms are situated in vacuum

and are irradiated by monoenegetic neutrons (the

second lower histogram); with (4) γ energy spectra

calculated by MCNP.

In Figure 3, Nunes et al.’s results show that

hydrogen contributed to the peak of prompt γ ray (2.2

MeV), carbon (4.9 MeV) and nitrogen (10.8 MeV).

Their results illustrate that the explosive spectra have

significant γ energy peaks which are the signatures

for C4 from 1 to 12 MeV (with 100 histogram bins

ranges from 1 – 12 MeV).

Obviously, a few major peaks are consistently

observed on the corresponding γ energy spectra

produced by GEANT4.8.2.p01 (Figure 3). Three of

the major peaks (~14600 counts of 2.2 MeV, ~130

counts of 4.9 MeV, ~720 counts of 10.8 MeV) match

with γ energy spectra produced by MCNP as stated

above. Other than the three major peaks, γ energy

spectra produced by carbon from inelastic interactions

(marked with *) and from s-wave captures include

1.85 MeV, 3.68 MeV, and *2.08 MeV. In addition,

nitrogen contributes *2.52 MeV, *2.90 MeV, *3.88

MeV, *4.50 MeV, 5.27 MeV, 5.30 MeV, *5.53 MeV,

*5.56 MeV, 6.32 MeV, 7.30 MeV, 8.31 MeV, 9.15

MeV, 9.93 MeV; whereas oxygen generates γ energy

spectrum with 3.05 MeV. All the γ energy spectra

above are in agreement with those published in

Ref. [19]. It is clear that γ energy spectra calculated

by GEANT4.8.2.p01 are discrete at 2.2 MeV and

above, whereas γ energy spectra below 2.2 MeV are

continuous. This is due to the various electromagnetic

processes, for instance, Bremsstrahlung, occur in this

energy range.

In Figure 4, a few extra γ energy spectra peaks

are recorded at 4.50 MeV, 5.27 MeV, 5.30 MeV, 5.53

MeV, 5.56 MeV, 6.32 MeV, 7.30 MeV, 9.15 MeV,

10.8 MeV in the top histogram compared to the other

two histograms below. These extra energy spectra

are similar with nitrogen peaks in Figure 3. These

background energy spectra may be contributed by the

atmospheric gas as the chemical composition setting

of rubber (C 5 H 8 ) does not contain nitrogen, besides,

nitrogen peaks do not appear in vacuum configuration

of these two experimental setups.

When C4 is occluded by rubber, the original

rubber’s prompt γ energy spectra will mix with

characteristic γ energy spectra from C4. This mixture

is clearly shown in Figure 5. High peak of the nitrogen

(10.8 MeV) and many extra γ energy spectra produced

Figure 4. Prompt γ energy spectra of Rubber (C5H8) generated by GEANT4.8.2.p01 compared with

MCNP.

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Journal of Science and Technology in the Tropics (2010) 6: 59-65


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Journal of Science and Technology in the Tropics (2010) 6: 59-65

by nitrogen indicates that the occluded material has

a high concentration of nitrogen. This indication

not only differentiates rubber with C4 from rubber

without C4, but also depicts the ratio of nitrogen in

each phantom in order to determine the presence of

plastic explosives [20].

Furthermore, Figure 3 and Figure 5 show that

incident neutrons with MB distribution energy will

only trigger slightly more extra γ energy spectra

compared to mono energetic neutrons. This outcome

is plausible as: (1) more types of elements in the

chemical composition and MB neutron energy

(more random energy) will give rise to more

possibilities of inelastic scattering; (2) although

the more random of the incident energy, the more

transitions incurred by inelastic scattering and

capture processes are expected, the difference

of randomized neutron energies is small. Hence,

small range of randomized neutron energy does not

impact much to the prompt γ energy spectra.

Figure 4 does not show an obvious difference of γ

energy spectra between MB and mono energetic.

Obviously, there are not many types of element in

the case of neutron-rubber scattering. The targets

of these simulations are in static form, Doppler

broadening does not affect the outcome of γ

energy spectra. Overall, the γ energy spectra

calculated by GEANT4.8.2.p01 shows good

agreement with MCNP in terms of the ratio of γ

energy spectra counts in histograms.

CONCLUSION

The present study on PGNAA computational

simulation which focuses on the comparison of

the corresponding characteristic γ rays is able to

show the differences among explosive and nonexplosive

occluded materials. Besides, we

demonstrate that γ rays generated from s-wave

neutron captured are not the only energy

spectrum to show the differences, non s-wave

neutron capture can also yield γ rays, which are

able to provide some significant energy peaks

for identifying hydrocarbon materials, e.g.

N (5.53 MeV) and N (5.56 MeV) which have

almost the same count as N (10.83 MeV) and

higher than C (4.95 MeV). Although the incident

neutron energies are randomly generated according

to Maxwell-Boltzmann distribution in mode 0.025

eV, the prompt γ spectra can still be able to show

Figure 5. Prompt γ energy spectrum of C4 hidden by Rubber generated by GEANT4.8.2.p01

compared with MCNP.

Jostt vol 6.indd 64 7/22/10 10:10:31 PM


the differences of γ energy spectrum for identifying

some hydrocarbon materials. The attempt of making

use of random incident neutron energy instead of

mono energetic neutron energy has paved the way

to imitate the physical condition in computational

simulation. For the time being, we consider GEANT4

should be a good alternative and candidate

for PGNAA simulation.

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Acknowledements – This work was supported in part by

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